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Ectonucleotidases: Some recent developments and a note on nomenclature
Abstract
Extracellular nucleotides such as ATP, ADP, UTP, UDP, and also diadenosine polyphosphates act as signaling molecules and can be inactivated by hydrolysis via ectonucleotidases. A considerable number of surface-located enzymes can potentially be involved in the extracellular hydrolysis pathway. These include the E-NTPDase family (ectonucleoside triphosphate diphosphohydrolase family), the E-NPP family (ectonucleotide pyrophosphatase/phosphodiesterase family), ecto-5′-nucleotidase, and alkaline phosphatases. In addition, activity of ectonucleoside diphosphokinase can interconvert extracellular nucleotides, and ATP can be used as a cosubstrate of ectoprotein kinase in the phosphorylation of surface-located proteins. Members of the various ectonucleotidase families reveal overlapping substrate specificity and tissue distribution whose functional significance needs to be further elucidated. Considerable progress has been made in the past several years in characterizing novel enzyme species and their molecular and functional properties. First knock-out mice reveal insight into physiological processes governed by the activity of specific ectonucleotidases. Together this work has led to a deeper understanding of the pathways of extracellular nucleotide metabolism, including their interplay with P2 and P1 receptors or also other physiological mechanisms. Drug Dev. Res. 52:44–56, 2001. © 2001 Wiley-Liss, Inc.
... A major challenge in delineating the functional roles of the P2Rs in vitro and in vivo is the rapid phosphate ester hydrolysis of both endogenous nucleotides and pharmacological tool compounds [9]. Ectonucleotidases are ubiquitous enzymes located on cell membrane surfaces and in circulating blood that rapidly dephosphorylate adenine and uridine nucleotides to adenosine and uridine, respectively [10]. These enzymes play a critical function in regulating the effects of nucleotides released from cells [11]. ...
... The susceptibility of purine and pyrimidine nucleotide P2Y or P2X agonists and antagonists to rapid dephosphorylation by ectonucleotidases has long been recognized [10,11]. Multiple ectonucleotidases and phosphatases, which are present at different levels in different cells and organs, are responsible for this metabolism [42,43]. ...
... At this time, however, there is not a consistent approach to scaling between in vitro methods to assess nucleotide stability and the actual in vivo pharmacokinetics of these compounds, further emphasizing the need for in vivo pharmacokinetic confirmation of the disposition of these nucleotide analogs. Unlike other drug-metabolizing enzyme families such as the cytochrome P450 family that are predominantly expressed in the liver, ectonucleotidases are an ubiquitous family of multiple enzymes that are not only expressed on the surface of every cell but circulated in the blood as well [10,11]. In the case of cytochrome P450-mediated metabolic clearance, techniques have been developed and summarized to predict in vivo intrinsic clearance of compounds from in vitro microsomal or hepatocyte stability data [60]. ...
Rapid phosphoester hydrolysis of endogenous purine and pyrimidine nucleotides has challenged the characterization of the role of P2 receptors in physiology and pathology. Nucleotide phosphoester stabilization has been pursued on a number of medicinal chemistry fronts. We investigated the in vitro and in vivo stability and pharmacokinetics of prototypical nucleotide P2Y1 receptor (P2Y1R) agonists and antagonists. These included the riboside nucleotide agonist 2-methylthio-ADP and antagonist MRS2179, as well as agonist MRS2365 and antagonist MRS2500 containing constrained (N)-methanocarba rings, which were previously reported to form nucleotides that are more slowly hydrolyzed at the α-phosphoester compared with the ribosides. In vitro incubations in mouse and human plasma and blood demonstrated the rapid hydrolysis of these compounds to nucleoside metabolites. This metabolism was inhibited by EDTA to chelate divalent cations required by ectonucleotidases for nucleotide hydrolysis. This rapid hydrolysis was confirmed in vivo in mouse pharmacokinetic studies that demonstrate that MRS2365 is a prodrug of the nucleoside metabolite AST-004 (MRS4322). Furthermore, we demonstrate that the nucleoside metabolites of MRS2365 and 2-methylthio-ADP are adenosine receptor (AR) agonists, notably at A3 and A1ARs. In vivo efficacy of MRS2365 in murine models of traumatic brain injury and stroke can be attributed to AR activation by its nucleoside metabolite AST-004, rather than P2Y1R activation. This research suggests the importance of reevaluation of previous in vitro and in vivo research of P2YRs and P2XRs as there is a potential that the pharmacology attributed to nucleotide agonists is due to AR activation by active nucleoside metabolites.
... The effects of nucleotides and nucleosides on purinergic receptors are regulated by the action of ectonucleotidases, which includes ectonucleoside triphosphate diphosphohydrolases (E-NTPDases), ectonucleoside pyrophosphatase/phosphodiesterases (E-NPPases), ecto-5 ¢ -nucleotidase/CD73 (Ecto-5 ¢ -NT/CD73) and alkaline phosphatases (ALP) Zimmermann 2001 . These enzymes operate in concert for the complete nucleotide hydrolysis (e.g. ...
... At present, the previously classi fi ed as E-type ATPases are identi fi ed as NTPDases belonging to the CD39 family. In mammals, at least eight related and homologous enzymes sharing fi ve apyraseconserved regions (ACRs), named NTDPase1 to 8, have been cloned and characterized: NTPDase1 (CD39, ATPDase, ecto-apyrase or ecto-ATP diphosphohydrolase), NTPDase2 (CD39L1, ecto-ATPase), NTPDase3 (CD39L3, HB6), NTPDase4 (UDPase, LALP70), NTPDase5 (CD39L4, ER-UDPase, PCPH), NTPDase6 (CD39L2), NTPDase7 (LALP1) and NTPDase8 Zimmermann 2001 ) . ...
... The opposite biological outcomes obtained by using NTPDase2 (high ATPase/ADPase ratio) and apyrase (low ATPase/ADPase ratio) as ATP/ADP scavengers, reveal a complex interactions between tumor, immune cells and purinergic mediators. This inverse effect could be due to the fact that whereas NTPDase1 hydrolyses ATP and ADP approximately equally well, the preferred degradation of ATP over ADP of NTPDase2 favors extracellular ADP accumulation Zimmermann 2001 ) . Considering that platelets express P2 receptors, which are activated by ADP (P2Y 1 and P2Y 12 ), we hypothesized that ADP produced by the NTPDase2 overexpressed in the implanted glioma cells could activate these receptors, leading to increased platelet recruitment and activation. ...
Among the pathological alterations that give tumor cells invasive potential, purinergic signaling is emerging as an important component. Studies performed in in vitro, in vivo and ex vivo glioma models indicate that alterations in the purinergic signaling are involved in the progression of these tumors. Gliomas have low expression of all E-NTPDases, when compared to astrocytes in culture. Nucleotides induce glioma proliferation and ATP, although potentially neurotoxic, does not evoke cytotoxic action on the majority of glioma cells in culture. The importance of extracellular ATP for glioma pathobiology was confirmed by the reduction in glioma tumor size by apyrase, which degrades extracellular ATP to AMP, and the striking increase in tumor size by over-expression of an ecto-enzyme that degrades ATP to ADP, suggesting the effect of extracellular ATP on the tumor growth depends on the nucleotide produced by its degradation. The participation of purinergic receptors on glioma progression, particularly P2X7, is involved in the resistance to ATP-induced cell death. Although more studies are necessary, the purinergic signaling, including ectonucleotidases and receptors, may be considered as future target for glioma pharmacological or gene therapy.
... and (d) ecto-5′-nucleotidases, which convert monophosphate nucleotides (e.g., AMP) to ADO. 115,116 In addition, the resulting ADO molecules can be further deaminated into inosine and, subsequently, hypoxanthine, by reactions mediated by adenosine deaminase (ADA) and purine nucleotide phosphorylase (PNP), respectively. 115,116 ...
... and (d) ecto-5′-nucleotidases, which convert monophosphate nucleotides (e.g., AMP) to ADO. 115,116 In addition, the resulting ADO molecules can be further deaminated into inosine and, subsequently, hypoxanthine, by reactions mediated by adenosine deaminase (ADA) and purine nucleotide phosphorylase (PNP), respectively. 115,116 ...
Since their discovery in the 1970s, purinergic receptors have been shown to play key roles in a wide variety of biologic systems and cell types. In the immune system , purinergic receptors participate in innate immunity and in the modulation of the adaptive immune response. In particular, P2 receptors, which respond to extra-cellular nucleotides, are widely expressed on leukocytes, causing the release of cytokines and chemokines and the formation of inflammatory mediators, and inducing phagocytosis, degranulation, and cell death. The activity of these receptors is regulated by ectonucleotidases-expressed in these same cell types-which regulate the
... Anchored to the cell membrane, these enzymes have the active site facing the extracellular medium, and also may be soluble in the interstitial medium. This group of proteins is comprised by the families of E-NTPDase, E-NPP, E-5 ′ -NT, and ALPs [25,72,77,78]. These ectonucleotidases are responsible for the hydrolysis of nucleotides to its respective nucleoside, constituting a highly efficient enzymatic cascade, being in charge of regulating the concentration and the time that these signaling molecules remain in the extracellular environment, and consequently stimulating their receptors [79]. ...
... P2X receptors are widely distributed in glial cells, neurons, and smooth muscle; they are linked to ion channels that, when activated, result in the opening of pores in the cell membrane leading to the passage of Na + , K + , and Ca 2+ ions [101][102][103][104][105][106]. P2Y are metabotropic receptors coupled to a G protein [107], and distributed in several tissues and systems, such as cardiac, nervous and vascular [15,78,108,109]. Additionally, P2X ionotropic receptors are further classified into seven subtypes from P2X1-7; on the other hand, P2Y metabotropic receptors can be further categorized into eight subtypes of receptors, namely P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , P2Y 13 , and P2Y 14 [107]. ...
The etiological agent of coronavirus disease (COVID-19) is the new member of the Coronaviridae family, a severe acute respiratory syndrome coronavirus 2 virus (SARS-CoV-2), responsible for the pandemic that is plaguing the world. The single-stranded RNA virus is capable of infecting the respiratory tract, by binding the spike (S) protein on its viral surface to receptors for the angiotensin II-converting enzyme (ACE2), highly expressed in the pulmonary tissue, enabling the interaction of the virus with alveolar epithelial cells promoting endocytosis and replication of viral material. The infection triggers the activation of the immune system, increased purinergic signaling, and the release of cytokines as a defense mechanism, but the response can become exaggerated and prompt the so-called “cytokine storm”, developing cases such as severe acute respiratory syndrome (SARS). This is characterized by fever, cough, and difficulty breathing, which can progress to pneumonia, failure of different organs and death. Thus, the present review aims to compile and correlate the mechanisms involved between the immune and purinergic systems with COVID-19, since the modulation of purinergic receptors, such as A2A, A2B, and P2X7 expressed by immune cells, seems to be effective as a promising therapy, to reduce the severity of the disease, as well as aid in the treatment of acute lung diseases and other cases of generalized inflammation.
... The levels of nucleotide are controlled by cell surface enzymes, known as ectonucleotidases [18]. The enzymes involved in this signaling cascade include the ectonucleoside triphosphate diphosphohydrolase (NTPDase), which hydrolyzes ATP to adenosine diphosphate (ADP) and/or adenosine monophosphate (AMP), followed by 5'-nucleotidase (5′-NT), which hydrolyzes AMP to adenosine, and nally, this molecule is deaminated to inosine by adenosine deaminase (ADA) [18,19]. ...
... The levels of nucleotide are controlled by cell surface enzymes, known as ectonucleotidases [18]. The enzymes involved in this signaling cascade include the ectonucleoside triphosphate diphosphohydrolase (NTPDase), which hydrolyzes ATP to adenosine diphosphate (ADP) and/or adenosine monophosphate (AMP), followed by 5'-nucleotidase (5′-NT), which hydrolyzes AMP to adenosine, and nally, this molecule is deaminated to inosine by adenosine deaminase (ADA) [18,19]. ...
Aluminum (Al) is considered a neurotoxic agent for biological systems and is recognized as a risk factor for neurodegenerative diseases. Al exposure occurs through the environment, water and human diet. The Al cationic form (Al ³⁺ ) has been associated with major deleterious effects on the central nervous system. The aim of this study was to investigate a possible mechanism that links Al and neurodegeneration in vivo . Here, we evaluated memory tests, purinergic signaling and inflammatory markers during long-term oral exposure to Al of mice in the total hippocampus. For this study, Swiss mice were divided into three groups: control (CT) group, AlCl 3 50 mg/kg group and AlCl 3 100 mg/kg group. The animals were orally treated with saline or Al ³⁺ at AlCl 3 form for 30 days. The memory parameters, body and brain weight, Al ³⁺ levels, DNA damage, enzyme activities, purinergic receptors and cytokine densities were assessed on the hippocampus of mice intoxicated with Al ³⁺ . Our results reveal that Al ³⁺ was able to reduce brain weight and accumulate in the hippocampus of animals treated with 100 mg/kg of salt. In addition, Al ³⁺ causes memory deficits and DNA damage. The adenosine triphosphate (ATP) hydrolysis was also affected by Al ³⁺ . Our results point to an increase in nucleoside triphosphate diphosphohydrolase (NTPDase), 5´-nucleotidase (5’-NT) and adenosine deaminase (ADA) activities. In addition, Al ³⁺ increases P2X7 and A2A receptor density, as IL-1β proinflammatory cytokine. Taken together, we suggest that Al ³⁺ can cause memory loss through DNA damage and alterations to the purinergic system through the proinflammatory process.
... In addition, a reduction in the O 2 consumption rate was observed for both fish species, suggesting the use of a common strategy when exposed to hypoxia. Furthermore, fish that are able to suppress metabolism when exposed to hypoxia can reduce their metabolic rates by 30% to 80% compared to standard metabolic rates (Van Ginneken et al., 1997, 2001Regan et al., 2017aRegan et al., , 2017b. Since this is not a complete cessation of the O 2 consumption rate, metabolic suppression can only prolong hypoxia survival, and not sustain it indefinitely (Regan et al., 2017a(Regan et al., , 2017b. ...
... The adenine nucleotides (ATP, ADP and AMP) and their derived nucleoside, Ado, act in the extracellular environment as signaling molecules (Zimmermann, 2001) involved in several physiological functions, such as: thromboregulation, homeostasis and platelet function (Zimmermann, 1999); in neurotransmission (Zimmermann, 2008) and adaptation to hypoxia (Schetinger et al., 1998). Recent study conducted by Baldissera et al. (2018) revealed that decreased NTPdase activity was not able to modulate the immune and inflammatory responses of Lophiosilurus alexandri exposed to hypoxia stress. ...
The Amazonian aquatic ecosystem undergoes seasonal variations and daily changes that directly affect the availability of oxygen. During the day the levels of oxygen can reach supersaturation, and at night can drop to zero. In this way, aquatic organisms are exposed daily to physiological challenges regarding the availability of oxygen. The present study revealed significant differences in the physiology and performance of two cichlids: Geophagus proximus (black water cichlid – from Negro River) and Chaetobranchopsis orbicularis (white water cichlid – from Amazon River), exposed to hypoxia. The white water cichlid showed lower value (1.99 ± 0.79 pKa) of critical pressure of oxygen (Pcrit) and a longer time (68.00 ± 14.11 min) for total loss of balance (LOE); however, this species showed 50% mortality during exposure to hypoxia, while the black water cichlid did not show mortality. Both cichlids presented a decrease in O2 consumption rate (OCR) during hypoxia.. In this sense, it was observed that the black water cichlid presented several physiological strategies during hypoxia, such as, a significant increase in plasma cortisol levels, nucleoside triphosphate diphosphohydrolase activity (for adenosine diphosphate (ADP) as a substrate) in the gills, and the activity of adenosine deaminase (ADA) in gills and liver, in addition to a significant increase in the activity of complexes (II-III) in the transporter chain of electrons in both analyzed tissues and succinate dehydrogenase activity of gills' mitochondria. On the other hand, the only physiological change observed in the white water cichlid was a significant reduction in the activity of complexes II-III in gills and liver. Based on our findings, we can hypothesize that the white water cichlid specie has less tolerant to hypoxia when compared to the black water cichlid.
... The formation of oligomers is essential for full catalytic activity. The biochemical properties of the E-NTPDases, their splice variants, and their tissue distribution have been reviewed in detail [1,19,20,[32][33][34][35][36]. ...
... The author of this article thus put together a nomenclature committee which finally met at the conference on "Ecto-ATPases and related ectonucleotidases" held in Diepenbeek, Belgium, in 1999 where it was agreed to apply a strictly biochemical enzyme nomenclature and to name this new protein family ectonucleoside triphosphate diphosphohydrolase family (E-NTPDase family) (EC: EC 3.6.1.5) and its individual members NTPDase1, NTPDase2, and so on [35,37]. While the name CD39 is frequently used for NTPDase1 in studies merely relating to its catalytic function, the author holds that the enzyme nomenclature should be applied. ...
Geoffrey Burnstock will be remembered as the scientist who set up an entirely new field of intercellular communication, signaling via nucleotides. The signaling cascades involved in purinergic signaling include intracellular storage of nucleotides, nucleotide release, extracellular hydrolysis, and the effect of the released compounds or their hydrolysis products on target tissues via specific receptor systems. In this context ectonucleotidases play several roles. They inactivate released and physiologically active nucleotides, produce physiologically active hydrolysis products, and facilitate nucleoside recycling. This review briefly highlights the development of our knowledge of two types of enzymes involved in extracellular nucleotide hydrolysis and thus purinergic signaling, the ectonucleoside triphosphate diphosphohydrolases, and ecto-5′-nucleotidase.
... glucose, adenosine) (Verkhratsky and Burnstock, 2014). This is also supported by studies showing that the use of P o is not limited by phosphatase activity in soil (Fransson and Jones, 2007;Zimmermann, 2001). The P cleavage process may occur indirectly as ectonucleotidases are primarily used for signal regulation involving extracellular nucleotides (e.g. ...
... The P cleavage process may occur indirectly as ectonucleotidases are primarily used for signal regulation involving extracellular nucleotides (e.g. ATP, ADP, UTP, and UDP; Zimmermann, 2001). We hypothesised, therefore, that any changes in phosphatase activity seen in soil under low P conditions maybe a plant-mediated rather than a microbially mediated response. ...
Maintaining an adequate phosphorus (P) supply for plants and microorganisms is central to agricultural production; however, the long-term effects of organic manure and inorganic fertilizer application on soil P cycling remain unclear. Organic P cycling in a sandy loam soil receiving medium and high rates of farmyard manure (FYM) and/or mineral fertilisers was studied in a long-term field experiment using a range of ¹⁴C/³³P isotope labelling approaches alongside metagenomic shotgun sequencing. FYM application alone negatively affected soil total P and organic P (Po) accumulation by enhancing crop offtake, enhancing Po mineralisation and stimulating P loss from the topsoil by reducing its P sorption potential. P mineralisation/immobilisation rate detected by the ³³P pool dilution method and the abundance of microbial P cycling genes were significantly correlated. Soil available C and N content was highly related to gross mineralisation/immobilisation rates and uptake/scavenging gene abundance. Microbial genes related to P uptake and metabolism were more abundant than P scavenging genes, while P scavenging genes may work efficiently as both of them can sustain similar P mineralisation and immobilisation rates. The addition of FYM also promoted phosphatase activity reflecting the increased supply of Po in these soils. Our study highlights that long-term FYM application regulates soil Po stocks and cycling, and that microbial functional gene abundance was coupled with P cycling rates.
... E-NTPDase is a family of enzymes that hydrolyses nucleoside tri-and diphosphate. These enzymes were formerly known as ecto-ATPases or ecto-apyrases [40,41]. Four of the eight NTPDase family members NTPDase1 (CD39), NTPDase2 (CD39L1), NTPDase3 (CD39L3), and NTPDase8 appear to be in charge of the majority of the nucleotide hydrolysis at the cell surface based on their membrane framework and distinctive catalytic characteristics [41]. ...
... The final version may differ from this version. NTPDase 1 is a divalent cation-dependent enzyme that catalyzes the dephosphorylation of nucleoside tri-and/or diphosphates at millimolar concentration of Mg 2+ at pH between 7 and 8 (Zimmermann, 2001). Previously, it has been reported that several nucleotidases, including NTPDase 1, 3, 4, and 8 are known to dephosphorylate ATP and UTP (Kukulski et al., 2005;Corriden et al., 2008;Zimmermann et al., 2012). ...
Gemcitabine (dFdC) and emtricitabine (FTC) are first-line drugs that are used for the treatment of pancreatic cancer and human immunodeficiency virus, respectively. The above drugs must undergo sequential phosphorylation to become pharmacologically active. Interindividual variability associated with the responses of the above drugs has been reported. The molecular mechanisms underlying the observed variability are yet to be elucidated. Although this could be multifactorial, nucleotidases may be involved in the dephosphorylation of drug metabolites due to their structural similarity to endogenous nucleosides. With these in mind, we performed in vitro assays using recombinant nucleotidases to assess their enzymatic activities toward the metabolites of dFdC and FTC. From the above in vitro experiments, we noticed the dephosphorylation of dFdC-monophosphate in the presence of two 5'-nucleotidases (5'-NTs), cytosolic 5'-nucleotidase IA (NT5C1A) and cytosolic 5'-nucleotidase III (NT5C3), individually. Interestingly, FTC monophosphate was dephosphorylated only in the presence of NT5C3 enzyme. Additionally, nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1) exhibited enzymatic activity toward both triphosphate metabolites of dFdC and FTC. Enzyme kinetic analysis further revealed Michaelis-Menten kinetics for both NT5C3-mediated dephosphorylation of monophosphate metabolites, as well as NTPDase 1-mediated dephosphorylation of triphosphate metabolites. Immunoblotting results confirmed the presence of NT5C3 and NTPDase 1 in both pancreatic and colorectal tissue that are target sites for dFdC and FTC treatment, respectively. Furthermore, sex-specific expression patterns of NT5C3 and NTPDase 1 were determined using mass spectrometry-based proteomics approach. Based on the above results, NT5C3 and NTPDase 1 may function in the control of the levels of dFdC and FTC metabolites. SIGNIFICANCE STATEMENT: Emtricitabine and gemcitabine are commonly used drugs for the treatment of human immunodeficiency virus and pancreatic cancer. To become pharmacologically active, both the above drugs must be phosphorylated. The variability in the responses of the above drugs can lead to poor clinical outcomes. Although the sources of drug metabolite concentration variability are multifactorial, it is vital to understand the role of nucleotidases in the tissue disposition of the above drug metabolites due to their structural similarities to endogenous nucleosides.
... Next, AMP is dephosphorylated into adenosine (ADO) by 5'-nucleotidase (CD73, EC 3.1.3.3.5). ADO bioavailability is controlled by adenosine deaminase (ADA, EC 3.5.4.4), which deaminates ADO into inosine (Zimmermann 2001;Colgan et al. 2006;Yegutkin 2008). ...
Metastatic melanoma is a very aggressive skin cancer. Platelets are constituents of the tumor microenvironment and, when activated, contribute to cancer progression, especially metastasis, and inflammation. P2Y12 is an adenosine diphosphate (ADP) receptor that triggers platelet activation. Inhibition of P2Y12 by clopidogrel bisulfate (CB) decreases platelet activation, which is also controlled by the extracellular concentration and the metabolism of purines by purinergic enzymes. We evaluated the effects of CB on the viability and proliferation of cultured B16-F10 cells. We also used a metastatic melanoma model with C57BL-6 mice to evaluate cancer development and purine metabolism modulation in platelets. B16-F10 cells were administered intraperitoneally to the mice. Two days later, the animals underwent a 12-day treatment with CB (30 mg/kg by gavage). We have found that CB reduced cell viability and proliferation in B16-F10 culture in 72h at concentrations above 30 μM. In vivo, CB decreased the tumor nodule counts and LDH levels and increased platelet purine metabolism. Our results showed that CB has significant effects on melanoma progression.
... Mutations in genes encoding CD39 and CD39L1 (ENTPD1 and ENTPD2, respectively) were previously reported as potential genetic causes involved in EIPH pathogenesis [8]. These enzymes, known as ENTPDase-1 and ENTPDase-2, respectively, are ectonucleotidases that hydrolyse nucleoside diphosphates and triphosphates and play a role in nucleotide metabolism related to haemostasis, such as nucleoside diphosphates (ADP) [9]. There is no difference between haemostatic variables related to coagulation and brinolysis in horses with and without EIPH. ...
Background
Exercise-induced pulmonary haemorrhage (EIPH) is characterized by the presence of blood from the lungs in the tracheobronchial tree after intense exercise in athletic horses. Despite the high prevalence of EIPH in horses, the primary aetiology remains unknown. Mutations in genes encoding CD39 and CD39L1 (ENTPD1 and ENTPD2, respectively) were previously reported as potential genetic causes involved in EIPH pathogenesis. However, the role these mutations associated with haemostatic functions is unknown.
Results
To investigate the association between EIPH and mutations (g.290A > G, g.291A > T, and g.338G > A) in the ENTPD1 and (g.646G > A) ENTPD2 genes, 76 Thoroughbred horses diagnosed with EIPH and 56 without clinical signs of EIPH (control group) by trachea-bronchial endoscopy were genotyped. The g.290A > G and g.338G > A mutations were linked, explaining why the same results were found in all horses. Both of these mutations were observed in 56.6% of homozygous and 39.5% of heterozygous EIPH horses. In the control group, 66.1% and 28.6% of horses were homozygous and heterozygous for these mutations, respectively. Approximately 2.6% (2/76) of EIPH horses and no control horses (0/56) were homozygous for the g.291A > T mutation. Meanwhile, in the EIPH group, there were 1.5% (1/68) homozygotes and 23.5% (16/58) heterozygotes for the g.464G > A mutation. In the control group, this mutation was observed only in heterozygotes (15.6%, 7/45). There was no significant difference between groups for any of the mutations.
Conclusions
The mutations previously described in the genes encoding CD39 and CD39L1 enzymes are highly present in the studied population. A possible breed predisposition may be involved; however, no association was found between the occurrence of EIPH and the presence of these mutations in the ENTPD1 and ENTPD2 genes in Thoroughbred horses in this study.
... ATP release mechanisms and degradation enzymes act on bacteria, and the first specific receptors for these molecules appeared in protozoa and eukaryotic unicellular algae [1]. The PSS nucleotides and adenosine are generated by the hydrolysis of ATP catalyzed by ectonucleotidase enzymes and released in the extracellular space [2]. Adenosine is a ubiquitous molecule involved in critical cellular processes, such as metabolism and cellular energy [3]. ...
Adenosine, a purine nucleoside with neuromodulatory actions, is part of the purinergic signaling system (PSS). Caenorhabditis elegans is a free-living nematode found in soil, used in biological research for its advantages as an alternative experimental model. Since there is a lack of evidence of adenosine’s direct actions and the PSS’s participation in this animal, such an investigation is necessary. In this research, we aimed to test the effects of acute and chronic adenosine at 1, 5, and 10 mM on nematode’s behaviors, morphology, survival after stress conditions, and on pathways related to the response to oxidative stress (DAF-16/FOXO and SKN-1) and genes products downstream these pathways (SOD-3, HSP-16.2, and GCS-1). Acute or chronic adenosine did not alter the worms’ morphology analyzed by the worms’ length, width, and area, nor interfered with reproductive behavior. On the other hand, acute and chronic adenosine modulated the defecation rate, pharyngeal pumping rate, and locomotion, in addition, to interacting with stress response pathways in C. elegans. Adenosine interfered in the speed and mobility of the worms analyzed. In addition, both acute and chronic adenosine presented modulatory effects on oxidative stress response signaling. Acute adenosine prevented the heat-induced-increase of DAF-16 activation and SOD-3 levels, while chronic adenosine per se induced DAF-16 activation and prevented heat-induced-increase of HSP-16.2 and SKN-1 levels. Together, these results indicate that exogenous adenosine has physiological and biochemical effects on C. elegans and describes possible purinergic signaling in worms.
... The ectonucleotidase families are the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase/CD39/NTP-Dase 1) and ectonucleotide pyrophosphatase/phosphodiesterases (E-NPP) that hydrolyze ATP and ADP to AMP. Plus, ecto-5-nucleotidase (E-5-nucleotidase/CD73) breaks down AMP into adenosine [24] and extracellular adenosine is converted into inosine by adenosine deaminase (ADA) [25]. ...
Type 2 diabetes mellitus (T2DM) is an important chronic disease around the world, and according to the World Health Organization, it is the 9th principal cause of global death. This pathology is characterized by high levels of circulating glucose as a result of insulin resistance, and it is well stated that inflammation related to obesity is directly associated with the development of the disease. The purinergic signalling is involved in both pancreatic destruction, which impairs insulin secretion, and the cytokine production that favors insulin resistance in T2DM. In this review, the purinergic signalling aspects will be discussed, showing the impact of the enzymes, nucleotides, nucleosides, and receptors of this system and the cytokines that result in inflammation, in the development and progression of T2DM, besides, pointing the purinergic receptors as a possible therapeutic approach.
... The catalytic role of ALP deals with the breakdown of ingested nucleotides, leading to inorganic phosphate and nucleosides. The latter may be further broken down by nucleosidases to produce purine and pyrimidine bases (Zimmermann, 2001). Once degraded, nucleosides and bases may be incorporated into liver, intestine, and skeletal muscle, although the mechanism whereby they exert their effects on tissue is unclear. ...
The present work aimed to investigate the effects of nucleotide oral administration on oxidative stress biomarkers, immune responses, gut morphology, serum biochemical parameters, and growth performance in calves from birth to 25 d of life. A total of 40 male Hol-stein Friesian calves were randomly divided in 2 groups. All the calves were born and reared on the same commercial dairy farm. They were fed the same colostrum, milk replacer, and calf starter. Five grams/head of an additive were orally administered with a syringe directly in the mouth to calves of the nucleotide group (NG). The additive contained 74.12 g/100 g of nucleic acids from hydrolyzed yeast, and 75.38% was free nucleotide sodium salt. The other group represented the negative control (CG). At 25 d of life all of the calves were slaughtered. Calves supplemented with nucleotides had a higher final live weight and improved average daily gain, which was associated with better efficiency of nutrient use. Oral nucleotide administration did not affect IgG absorption efficiency; however, NG calves showed greater duodenum villi length and higher crypt depth compared with CG. Oral nucleotide administration increased the activity of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) and the antioxidant capacity [ferric reducing antioxidant power and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) scavenging activity] both in plasma and in liver. An enhanced ability of cells to counter reactive oxygen species-and reactive nitrogen species-mediated damage was also observed in peripheral blood mononuclear cells from NG. The findings highlight the effectiveness of oral nucleotide administration, and potentially dietary supplementation of nucleotides, in boosting oxidative and immune status in newborn calves.
... Purinergic receptors activate the binding of signals to their corresponding receptors, primarily to ATP nucleotides. Under physiological conditions, the extracellular ATP is released from cells and extracellular ATP levels are regulated by ectonucleotidases, which catalyze their degradation (Zimmermann, 2001). P2X receptor subtypes each have phenotype-dependent structures, ionic characteristics, and responsiveness to various allosteric modulators, agonists, and antagonists (Li et al. 2009). ...
The primary therapy for symptomatic motor benefits linked to patients diagnosed with Parkinson’s Disease (PD) has been levodopa (L-Dopa). L-DOPA is a precursor of dopamine (DA) and represents the gold-standard pharmacological treatment for Parkinson's disease. Nevertheless, L- DOPA has had numerous drawbacks in long-term treatment, leading to motor problems as the therapy becomes less effective. Presently there is no cure for PD, where the disease has been linked to a dopaminergic disorder that affects the nigrostriatal pathway triggered by dopamine deficiency. Purinergic ionotropic P2X4 receptors (P2X4s) have been introduced as a protein candidate that would regulate dopamine activity. P2X4s are critical dopamine activity modulations that increase the release of calcium into neurons, leading to an increase in secretion of DA. The goal of my thesis project is to illustrate the interaction between ivermectin (IVM) modulation of P2X4s using PD models, 6-OHDA and MPTP to evaluate the effect of P2X4s that are involved in alterations in motor behavior for PD. This will be accomplished by summarizing drug efficacy via the concept of IVM's therapeutic targets with the presence of L-DOPA on P2X4s. Overall, this work should help in the identification of potential therapeutic roles of the purinergic receptor system in the activation of neurotransmission of DA and the sex differences in the response by IVM in C57BL/6J mice.
... Adenosine is a well-established factor in suppressing immune responses [12]. Ectonucleotidases are cell membrane enzymes that can cleave the extracellular nucleotides to form nucleosides [13]. Ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1), also referred to as CD39 and NTPDase, can substantially cleave ATP and uridine triphosphate (UTP) into ADP and uridine diphosphate (UDP). ...
The immunosuppressive tumor microenvironment has been implicated in attenuating anti-tumoral immune responses and tumor growth in various cancers. Inhibitory immune checkpoints have been introduced as the primary culprits for developing the immunosuppressive tumor microenvironment. Therefore, a better understanding of the cross-talk between inhibitory immune checkpoints in the tumor microenvironment can pave the way for introducing novel approaches for treating affected patients. Growing evidence indicates that CD39 and CD73, as novel checkpoints, can transform adenosine triphosphate (ATP)-mediated pro-inflammatory tumor microenvironment into an adenosine-mediated immunosuppressive one via the purinergic signaling pathway. Indeed, enzymatic processes of CD39 and CD73 have crucial roles in adjusting the extent, intensity, and chemical properties of purinergic signals. This study aims to review the biological function of CD39 and CD73 and shed light on their significance in regulating anti-tumoral immune responses in various cancers.
... Extracellular ATP can be directly degraded to AMP and pyrophosphate (PPi) by ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (ENPP1) or to ADP by ENPP3 [38]. Interestingly, membrane-bound tissue nonspecific alkaline phosphatase (TNAP), which belongs to the alkaline phosphatase family and is mainly involved in mechanisms controlling normal skeletal mineralization and pathophysiological abnormalities, subsequently hydrolyzes PPi to inorganic phosphate [39]. ...
Bone is one of the major tissues that undergoes continuous remodeling throughout life, thus ensuring both organic body growth during development and protection of internal organs as well as repair of trauma during adulthood. Many endogenous substances contribute to bone homeostasis, including purines. Their role has increasingly emerged in recent decades as compounds which, by interacting with specific receptors, can help determine adequate responses of bone cells to physiological or pathological stimuli. Equally, it is recognized that the activity of purines is closely dependent on their interconversion or metabolic degradation ensured by a series of enzymes present at extracellular level as predominantly bound to the cell membrane or, also, as soluble isoforms. While the effects of purines mediated by their receptor interactions have sufficiently, even though not entirely, been characterized in many tissues including bone, those promoted by the extracellular enzymes providing for purine metabolism have not been. In this review, we will try to circumstantiate the presence and the role of these enzymes in bone to define their close relationship with purine activities in maintaining bone homeostasis in normal or pathological conditions.
... NTPDases 4, 5, 6, and 7 are localized in the intracellular space where they are anchored to intracellular organelle membranes or vesicles and have lower affinity to nucleotides when compared with extracellular ones (37) (Fig. 1). NTPDases 5 and 6 are anchored to the organelles by one transmembrane domain, whereas NTPDases 4 and 7 are anchored to the organelles by two transmembrane domains (38,39). ...
Nucleoside triphosphate diphosphohydrolases (NTPDases) are a family of enzymes that hydrolyze nucleotides such as ATP, UTP, ADP, and UDP to monophosphates derivates such as AMP and UMP. The NTPDase family consists of eight enzymes, of which NTPDases 1, 2, 3, and 8 are expressed on cell membranes thereby hydrolyzing extracellular nucleotides. Cell membrane NTPDases are expressed in all tissues, in which they regulate essential physiological tissue functions such as development, blood flow, hormone secretion, and neurotransmitter release. They do so by modulating nucleotide-mediated purinergic signaling through P2 purinergic receptors. NTPDases 1, 2, 3, and 8 also play a key role during infection, inflammation, injury, and cancer. Under these conditions, NTPDases can contribute and control the pathophysiology of infectious, inflammatory diseases and cancer. In this review, we discuss the role of NTPDases, focusing on the less understood NTPDases 2-8, in regulating inflammation and immunity during infectious, inflammatory diseases, and cancer.
... Purinergic signaling is inactivated through nucleotide metabolization, performed by a multienzymatic complex composed of ectonucleotidases. These enzymes are located on the cell surface and may also be found in the interstitial medium or within body fluids; they exhibit broad substrate specificity and tissue distribution [4]. Nucleotide hydrolysis includes a role for ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases), ecto-nucleotide pyrophosphatase/phosphodiesterases (E-NPPs), ecto-5'-nucleotidases, and alkaline phosphatases [5]. ...
... Ecto-nucleotidases are group of nucleotide hydrolyzing enzymes that are mainly involved in the hydrolysis of nucleotides such as nucleoside 5′-triphosphate, 5′-diphosphate and 5′-monophosphate [1]. Primarily, they are comprises of four major classes i.e., the ectonucleoside triphosphate diphosphohydrolases (eNTPDases), ecto-5′-nucleotidase (CD73), ectonucleotide pyrophosphatase/phosphodiesterase (eNPPs), and alkaline phosphatases (APs). ...
Aims:
The present study was conducted to examine the inhibitory effects of synthesized sulfonylhydrazones on the expression of CD73 (ecto-5'-NT).
Background:
CD73 (ecto-5'-NT) represents the most significant class of ecto-nucleotidases which are mainly responsible for dephosphorylation of adenosine monophosphate to adenosine. Inhibition of CD73 played an important role in the treatment of cancer, autoimmune disorders, precancerous syndromes, and some other diseases associated with CD73 activity.
Objective:
Keeping in view the significance of CD73 inhibitor in the treatment of cervical cancer, a series of sulfonylhydrazones (3a-3i) derivatives synthesized from 3-formylchromones were evaluated.
Methods:
All sulfonylhydrazones (3a-3i) were evaluated for their inhibitory activity towards CD73 (ecto-5'-NT) by the malachite green assay and their cytotoxic effect was investigated on HeLa cell line using MTT assay. Secondly, most potent compound was selected for cell apoptosis, immunofluorescence staining and cell cycle analysis. After that, CD73 mRNA and protein expression were analyzed by real-time PCR and Western blot.
Results:
Among all compounds, 3h, 3e, 3b, and 3c were found the most active against rat-ecto-5'-NT (CD73) enzyme with IC50 (µM) values of 0.70 ± 0.06 µM, 0.87 ± 0.05 µM, 0.39 ± 0.02 µM and 0.33 ± 0.03 µM, respectively. These derivatives were further evaluated for their cytotoxic potential against cancer cell line (HeLa). Compound 3h and 3c showed the cytotoxicity at IC50 value of 30.20 ± 3.11 µM and 86.02 ± 7.11 µM, respectively. Furthermore, compound 3h was selected for cell apoptosis, immunofluorescence staining and cell cycle analysis which showed promising apoptotic effect in HeLa cells. Additionally, compound 3h was further investigated for its effect on expression of CD73 using qRT-PCR and western blot.
Conclusion:
Among all synthesized compounds (3a-3i), Compound 3h (E)-N'-((6-ethyl-4-oxo-4H-chromen-3-yl) methylene)-4-methylbenzenesulfonohydrazide was identified as most potent compound. Additional expression studies conducted on HeLa cell line proved that this compound successfully decreased the expression level of CD73 and thus inhibiting the growth and proliferation of cancer cells.
... NTPDase3 and -8 have intermediate preference for ATP, causing a slight accumulation of diphosphonucleosides. In contrast, NTPDase5 and -6 present preference for the hydrolysis of nucleoside diphosphates [12], while ecto-5′nucleotidase (CD73) is responsible for the degradation of AMP to its respective nucleoside, adenosine [13]. ...
Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm, characterized by the occurrence of the t(9;22)(q34;q11) translocation. First-line therapy for CML consists of treatment with imatinib mesylate, which selectively inhibits the BCR-ABL protein by competing for its ATP-binding site. Adenine nucleotide signaling is modulated by the ectonucleotidases and this pathway is related to tumorigenic processes. Considering the relationship between ATP and cancer, we aimed to evaluate the influence of imatinib mesylate on the expressions and functions of the NTPDase and ecto-5′-nucleotidase (CD73) enzymes in imatinib-sensitive and -resistant K-562 cell lines. mRNA analysis showed that K-562 cells express all ENTPDs and NT5E. However, when treated with imatinib mesylate for 24 h, the expression of ENTPD1, -2, -3 and -5 increased, leading to a higher nucleotides hydrolysis rate. HPLC analysis identified increased ATP degradation in cells after 24 h of treatment, with consequent ADP and AMP formation, corroborating the increase in gene and protein expression of ectonucleotidases as observed in previous results. On the other hand, we observed that imatinib-resistant K-562 cells presented a decrease in nucleotide hydrolysis and expressions of ENTPD1 and -5. These results suggest an involvement of imatinib in modulating ectonucleotidases in CML that will need further investigation. Since these ectonucleotidases have important catalytic activities in the tumor microenvironment, their modulation in CML cells may represent an important therapeutic approach to regulate levels of extracellular adenine nucleotides.
... In addition, these enzymes differ in their ratios and preferably for the substrate hydrolysis. The NTPDase 1 hydrolyzes ATP and ADP in the same way; also, NTPDases 2, 3 and 8 hydrolyze ATP more than ADP, and NTPDase 4 preferentially hydrolyzes UDP (Zimmermann, 2001). Based on these findings, we believe that the effects found in the hippocampus and cerebral cortex can be related to the activity of different enzyme isoforms. ...
Aims: We investigated whether the treatment with anthocyanins prevents the scopolamine-induced memory deficits and whether ectonucleotidase activities and purine levels are altered in the cerebral cortex (CC) and hippocampus (HC) in this model of mnemonic deficit in rats. Main methods: The animals were divided into 4 experimental groups: control (vehicle), anthocyanins (Antho), scopolamine (SCO), and scopolamine plus anthocyanins (SCO+Antho). After seven days of treatment, they were tested in the inhibitory avoidance task and open field test and submitted to euthanasia. The CC and the HC were collected for biochemical assays. The effect of treatment with Antho (200 mg kg −1 , i.p.) was investigated in rats trained to a stable level of performance and post-treated with SCO (1 mg kg −1 , i.p. 30 min after training). Key findings: The treatment with SCO decreased the step-down latency in inhibitory avoidance task. Antho prevented the scopolamine-induced memory impairment and also the increase of NTPDase activity in the CC and HC. Furthermore, the treatment with anthocyanins prevents the decrease in 5′-nucleotidase activity and the increase in adenosine deaminase activity induced by SCO in HC. In addition, the treatment with Antho prevented the decrease in ATP levels induced by SCO in the CC and HC. Significance: Our results show that scopolamine may affect purinergic enzymatic cascade or cause alterations in energy metabolism inducing loss of memory. In contrast Antho could reverse these changes, suggesting a neuroprotective effect of Antho on ectonucleotidase activities and neuronal energetic metabolism.
... Many members of this family are membrane glycoproteins whose catalytic domain faces either the extracellular medium or the lumen of intracellular organelles, such as the Golgi apparatus [138,139]. Ynd1 is inserted in the Golgi membrane such that its N-terminal 500 residues containing its catalytic domain are situated in the Golgi lumen and its C-terminal 113 residues face the cytosol [140,141]. Ynd1 and the PP2A regulatory subunit Cdc55 were found to have an additive contribution to E4orf4-induced toxicity in yeast. However, the two proteins physically interacted, and a functional interaction was also observed as deletion of Ynd1 sensitized the cells to E4orf4-induced toxicity in the presence of overexpressed Cdc55 [137]. ...
The adenovirus (Ad) E4orf4 protein is a small 14 kDa polypeptide endowed with important viral regulatory functions. Although deletion of E4orf4 does not have a major effect on Ad replication due to redundancy among many Ad proteins, E4orf4 provides several functions that improve viral replication. E4orf4 contributes to temporal regulation of virus infection by down‐regulating early viral gene expression and by altering splicing patterns of Ad mRNAs. It also optimizes the cellular environment for Ad replication by activating the mTOR pathway to increase viral protein production and by impacting the cell cycle. In addition, E4orf4 participates in the inhibition of the host DNA damage response, promoting the ability of Ad to counteract this antiviral defense mechanism. To fulfil these functions, E4orf4 interacts with numerous cellular proteins, including the major E4orf4 partner, protein phosphatase 2A (PP2A). When expressed alone, outside the context of virus infection, E4orf4 induces an evolutionarily‐conserved, caspase‐independent, cancer‐selective cell death with many interesting characteristics. This review critically describes E4orf4’s contribution to Ad infection and cancer‐cell death.
... Moreover, CD73 is a lymphocyte surface protein, used as a marker of B and T lymphocyte maturation. It is the main responsible for the extracellular nucleotide hydrolysis into Ado (Zimmermann, 2001). Several studies have demonstrated that these ectoenzymes act in coordinating the regulation of immune responses in diseases and are related with the development and progression of chronic disorders de Souza Cardoso et al., 2018;Do Carmo Araújo et al., 2005;Martins et al., 2016;Spanevello et al., 2009;Zanini et al., 2012). ...
... In this study, nine APY homologs were identified in the wheat genome (Fig. 1), and were further divided into three groups based on their phylogenetic relationship and three-dimensional structures (Fig. 4). It was postulated that the transmembrane character could be associated with the subcellular locations of the proteins (Chiu et al., 2012;Knowles, 2011;Zimmermann, 2010). In mammals, all four ecto-APYs contained both N-and C-terminal transmembrane domains, while others were endo-APYs with only C-terminal transmembrane domains (Knowles, 2011;Tsan-Yu et al., 2015). ...
APYRASE s, which directly regulate intra- and extra-cellular ATP homeostasis, play a pivotal role in the regulation of various stress adaptations in mammals, bacteria and plants. In the present study, we identified and characterized wheat APYRASE family members at the genomic level in wheat. The results identified a total of nine APY homologs with conserved ACR domains. The sequence alignments, phylogenetic relations and conserved motifs of wheat APYs were bioinformatically analyzed. Although they share highly conserved secondary and tertiary structures, the wheat APYs could be mainly categorized into three groups, according to phylogenetic and structural analysis. Additionally, these APYs exhibited similar expression patterns in the root and shoot, among which TaAPY3-1 , TaAPY3-3 and TaAPY3-4 had the highest expression levels. The time-course expression patterns of the eight APY s in response to biotic and abiotic stress in the wheat seedlings were also investigated. TaAPY3-2 , TaAPY3-3 , TaAPY3-4 and TaAPY6 exhibited strong sensitivity to all kinds of stresses in the leaves. Some APY s showed specific expression responses, such as TaAPY6 to heavy metal stress, and TaAPY7 to heat and salt stress. These results suggest that the stress-inducible APY s could have potential roles in the regulation of environmental stress adaptations. Moreover, the catalytic activity of TaAPY3-1 was further analyzed in the in vitro system. The results showed that TaAPY3-1 protein exhibited high catalytic activity in the degradation of ATP and ADP, but with low activity in degradation of TTP and GTP. It also has an extensive range of temperature adaptability, but preferred relatively acidic pH conditions. In this study, the genome-wide identification and characterization of APY s in wheat were suggested to be useful for further genetic modifications in the generation of high-stress-tolerant wheat cultivars.
... These ecto-NTPDases hydrolyze nucleosides di-and triphosphate with different substrate preferences (6) and are involved in metabolism and control of extracellular nucleotide/nucleoside concentrations, which result in the modulation of purinergic signaling (7). Ecto-5'-nucleotidase hydrolyzes nucleotide monophosphates such as AMP to the respective nucleosides and is a key enzyme in the nucleotide degradation pathway (8)(9)(10). It helps to maintain strict control of adenosine levels, and its activity is most prominent in glia (11). ...
Extracellular nucleotides are degraded by several ecto-enzymes present on the cell membrane, and also by soluble enzymes in the bloodstream. Previous work has already found elevated 5'-nucleotidase activity in the serum of patients with head and neck cancer, but very few reports are available on the involvement of the nucleotidases in brain tumor development. The aim of this study was to evaluate the hydrolysis of adenine nucleotides in the blood serum of brain tumor patients. Blood samples were obtained from 15 patients between 17 and 76 years old with brain tumors, and from 25 healthy individuals (control). The pathological status of each tumor was confirmed by histology. Brain tumors included in this study were high-grade gliomas (glioblastoma and anaplastic astrocytoma) and low-grade gliomas (low-grade astrocytoma and meningioma). For the estimation of nucleotidase activity we used ATP (1.5mM), ADP (1.5mM) and AMP (2.0mM) as substrates. The products of enzymatic analysis were measured by spectrophotometer, and the amount of inorganic phosphate (Pi) liberated was determined by the method of malachite green. The results showed that, in comparison with controls, AMPase activity was significantly increased (p<0.05) in high-grade intracranial neoplasms, while there were no significant differences in ATPase and ADPase activities. In conclusion, our results indicate that 5'-NT activity may be useful for monitoring patients with high-grade brain tumors. We have reported the development of a reliable method to detect the activities of nucleotidases in the serum of glioma patients. Aumento da atividade da 5'nucleotidase no soro de pacientes com tumor cerebral RESUMO-Os nucleotídeos extracelulares são degradados por várias ectoenzimas que estão presentes na membrana celular e também por enzimas solúveis presentes na corrente sanguínea. Trabalhos anteriores já encontraram elevada atividade da 5'-nucleotidase no soro de pacientes com câncer de cabeça e pescoço, mas poucos relatos estão disponíveis sobre o envolvimento dos nucleotidases no desenvolvimento de tumor cerebral. O objetivo deste estudo foi avaliar a hidrólise de nucleotídeos da adenina no soro de pacientes com tumor cerebral. As amostras de sangue foram obtidas de 15 pacientes entre 17 e 76 anos com tumores cerebrais e de 25 indivíduos saudáveis (controle). O estado patológico de cada tumor foi confirmado por técnica histológica. Os tumores cerebrais incluídos neste estudo foram gliomas de alto grau (glioblastoma e astrocitoma anaplásico) e gliomas de baixo grau (astrocitoma de baixo grau e meningioma). Para a estimativa da atividade da nucleotidase foi utilizado como substratos: ATP (1,5mM), ADP (1,5mM) e AMP (2,0mM). Os produtos da análise enzimática foram medidos com um espectrofotômetro e a quantidade de fosfato inorgânico (Pi) liberado foi determinada pelo método de verde de malaquita. Os resultados mostraram que a atividade da AMPase foi significativamente aumentada (p<0,05) em neoplasias intracranianas de alto grau, enquanto que não houve diferenças significativas na atividade da ATPase e da ADPase, em comparação com os controles. Em conclusão, nossos resultados indicam que a atividade da 5'-nucleotidase pode ser útil P. Engroff et al. / Vittalle 28 (2016) 103-110. 103 para monitorar pacientes com tumores cerebrais de alto grau. Também relatamos o desenvolvimento de um método confiável para detectar as atividades das nucleotidases no soro de pacientes com glioma.
In this study various of thieno[3,2‐d]pyrimidine derivatives have been synthesized by treating different secondary amines through aromatic nucleophilic substitution reaction (SNAr) followed by Suzuki reaction with aryl and heteroaryl boronic acids. A bis‐Suzuki coupling was also performed to generate bis‐aryl thienopyrimidine derivatives. The synthesized compounds were screened for the hydrolytic activity of h‐NTPdase1, h‐NTPdase2, h‐NTPdase3, and h‐NTPdase8. The compound N‐benzyl‐N‐methyl‐7‐phenylthieno[3,2‐d]pyrimidin‐4‐amine 3 j selectively inhibits the activity of h‐NTPdase1 with IC50 value of 0.62±0.02 μM whereas, the compound 4 d was the most potent inhibitor of h‐NTPdase2 with sub‐micromolar IC50 value of 0.33±0.09 μM. Similarly, compounds 4 c and 3 b were found to be selective inhibitors for isozymes h‐NTPdase3 (IC50=0.13±0.06 μM) and h‐NTPdase8 (IC50=0.32±0.10 μM), respectively. The molecular docking study of the compounds with the highest potency and selectivity revealed the interactions with the important amino acid residues.
The present study evaluated the effect of caffeic acid (CA) on behavioral learning and memory tasks in the diabetic state. We also evaluated the effect of this phenolic acid on the enzymatic activities of acetylcholinesterase (AChE), ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), ecto-5-nucleotidase (E-5'-NT) and adenosine deaminase (ADA) as well as on the density of M1R, α7nAChR, P2×7R, A1R, A2AR and inflammatory parameters in the cortex and hippocampus of diabetic rats. Diabetes was induced by a single intraperitoneal dose of streptozotocin (STZ, 55 mg/kg). The animals were divided into six groups: control/vehicle; control/CA 10 and 50 mg/kg; diabetic/vehicle; diabetic/CA 10 and 50 mg/kg, treated by gavage. The results showed that CA improved learning and memory deficits in diabetic rats. Also, CA reversed the increase in AChE and ADA activities and reduced ATP and ADP hydrolysis. Moreover, CA increased the density of M1R, α7nAChR, and A1R receptors and reversed the increase in P2×7R and A2AR density in both evaluated structures. In addition, CA treatment attenuated the increase in NLRP3, caspase 1, and IL-1β density in the diabetic state; moreover, it increased the density of IL-10 in the diabetic/CA 10 mg/kg group. The results indicated that CA treatment positively modified the activities of cholinergic and purinergic enzymes and the density of receptors, and improved the inflammatory parameters of diabetic animals. Thus, the outcomes suggest that this phenolic acid could improve the cognitive deficit linked to cholinergic and purinergic signaling in the diabetic state.
The therapeutic potential of purinergic signaling has been explored for a wide variety of diseases, including those related to the skin. In this study, we used the self-assembled skin substitutes (SASS), a highly functional reconstructed human skin model, which shares many properties with normal human skin, to study the impact of purinergic receptors agonists, such as ATP, UTP and a P2Y receptor antagonist, Reactive Blue 2 during wound healing. After treating the wounded skins, we evaluated the wound area, reepithelialization, length of migrating tongues toward the wound, quality of the skins through the cytokeratin 10 and laminin-5 expression, epidermal and dermal cell proliferation. In addition, the expression of the main ectoenzymes capable of hydrolyzing nucleotides were investigated through the wounded SASS regions: unwounded region, wound margin, intermediate region and migrating epidermal tongue. After 3 days, under the UTP treatment, the wounded SASS showed an increase in the reepithelialization and in the proliferation of keratinocytes and fibroblasts, without altering the quality of the skin. We also identified the presence of the ectoenzymes NTPDase1 and NPP1 in the reconstructed human skin model, suggesting their involvement in wound healing. Considering the need for new therapies capable of promoting healing in complex wounds, although these results are still preliminary, they suggest the involvement of extracellular nucleotides in human skin healing and the importance to understand their role in this mechanism. New experiments it will be necessary to determine the mechanisms by which the purinergic signaling is involved in the skin wound healing.
The human epidermal melanocyte (hEM) are melanin-producing cells that provide skin pigmentation and protection against ultraviolet radiation. Although purinergic signaling is involved in skin biology and pathology, the presence of NTPDase members, as well as the rate of nucleotides degradation by melanocytes was not described yet. Therefore, in this study, we analyzed the expression of ectonucleotidases in hEM derived from discarded foreskin of male patients. The expression of purinergic enzymes was confirmed by mRNA and flow cytometry. Among the ectonucleotidases, NTPDase1 and CD73 were the ectoenzymes with higher expression. The hydrolysis rate for ATP, ADP and AMP was low, in comparison to other primary cells already investigated. The amount of ATP in the culture medium was increased after a scratch wound, decreasing to basal levels in 48h, while the NTPDase1 and P2X7 expression increased. Therefore, it is possible to suggest that after cell injury, the ATP released by hEM into the extracellular space will be hydrolyzed by ectonucleotidases as the NTPDase1 that will control the levels of nucleotides in the skin microenvironment.
Ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) are enzymes located on the surface of the T. cruzi plasma membrane, which hydrolyze a wide range of tri-/-diphosphate nucleosides. In this work, we used previously developed genetically modified strains of Trypanosoma cruzi (T. cruzi), hemi-knockout (KO +/−) and overexpressing (OE) the TcNTPDase-1 gene to evaluate the parasite infectivity profile in a mouse model of acute infection (n = 6 mice per group). Our results showed significantly higher parasitemia and mortality, and lower weight in animals infected with parasites OE TcNTPDase-1, as compared to the infection with the wild type (WT) parasites. On the other hand, animals infected with (KO +/−) parasites showed no mortality during the 30-day trial and mouse weight was more similar to the non-infected (NI) animals. In addition, they had low parasitemia (45.7 times lower) when compared with parasites overexpressing TcNTPDase-1 from the hemi-knockout (OE KO +/−) group. The hearts of animals infected with the OE KO +/− and OE parasites showed significantly larger regions of cardiac inflammation than those infected with the WT parasites (p < 0.001). Only animals infected with KO +/− did not show individual electrocardiographic changes during the period of experimentation. Together, our results expand the knowledge on the role of NTPDases in T. cruzi infectivity, reenforcing the potential of this enzyme as a chemotherapy target to treat Chagas disease (CD).
Embora existam diversos grupos brasileiros de pesquisa da sinalização purinérgica, o conhecimento sobre o tema tem sido amplamente difundido em língua inglesa em periódicos internacionais. Em língua portuguesa, os estudos, de um modo geral, constituem dissertações e teses. Todos os colaboradores do livro são pesquisadores brasileiros que estudam alguma situação fisiológica ou patológica envolvendo o sistema purinérgico. O livro trata do funcionamento da sinalização purinérgica em condições fisiológicas gerais e sobre a história das enzimas e dos receptores purinérgicos. Aborda situações fisiológicas, como a modulação do sistema purinérgico pelo exercício físico e por moléculas nutracêuticas. E dedica alguns capítulos para a relação da sinalização purinérgica em condições patológicas: diversos tipos de câncer; doenças endócrinas, como diabetes e disfunções da glândula tireoide; doença renal; hipertensão arterial sistêmica; doenças degenerativas, como doença de Alzheimer e doença macular relacionada à idade; doenças parasitárias e virais, dentre outras.
The role of the purinergic signal has been extensively investigated in many tissues and related organs, including the central and peripheral nervous systems as well as the gastrointestinal, cardiovascular, respiratory, renal, and immune systems. Less attention has been paid to the influence of purines in the oral cavity, which is the first part of the digestive apparatus and also acts as the body’s first antimicrobial barrier. In this review, evidence is provided of the presence and possible physiological role of the purinergic system in the different structures forming the oral cavity including teeth, tongue, hard palate, and soft palate with their annexes such as taste buds, salivary glands, and nervous fibers innervating the oral structures. We also report findings on the involvement of the purinergic signal in pathological conditions affecting the oral apparatus such as Sjögren’s syndrome or following irradiation for the treatment of head and neck cancer, and the use of experimental drugs interfering with the purine system to improve bone healing after damage. Further investigations are required to translate the results obtained so far into the clinical setting in order to pave the way for a wider application of purine-based treatments in oral diseases.
Introduction:
Nucleoside triphosphate diphosphohydrolases (NTPDases), alkaline phosphatases (APs), and ecto-nucleotide pyrophosphatases/phosphodiesterases (NPPs) are nucleotidases found on the cell surface. It is a promising therapeutic target for a range of disorders, including fibrosis, tumour metastasis, pruritus, inflammation, multiple sclerosis, and autoimmune diseases. As a result, therapeutic intervention including selective inhibitors of NPPs is required.
Area covered:
Many chemical substances, including pyrazole, pyridine and bicyclic compounds have demonstrated promising inhibitory potential for ecto-nucleotide pyrophosphatase/phosphodiesterases. The chemistry and clinical applications of NPPs inhibitors patented between 2015 and 2020 are discussed in this review.
Expert opinion:
: In recent years, there has been a lot of effort put into finding effective and selective inhibitors of NPPs. Despite the fact that a variety of synthetic inhibitors have been created, only a few investigations on their in vivo activity have been published. In addition to IOA-289 which has passed Phase Ia clinical trials; potent ATX inhibitor compounds such as BLD-0409, IPF and BBT-877 have been placed in phase I clinical studies. Some of the most promising ATX inhibitors in recent years are closely related analogs of previously known inhibitors, such as PF-8380. Knowledge of the structure activity relationship of such promising inhibitors can potentially translate into the discovery of more potent and effective inhibitors of NPP with a variety of structural characteristics and favourable therapeutic activities.
Background
Phosphodiesterases (PDEs) are a wide group of enzymes with multiple therapeutic actions, including vasorelaxation, cardiotonic, antidepressant, anti-inflammatory, antithrombotic, anti-spasmolytic, memory-enhancing, and anti-asthmatic. PDEs with eleven subtypes from PDE-1 to PDE-11 typically catalyze the cleavage of the phosphodiester bond and, hence, degrades either cyclic adenosine monophosphate (cAMP) or cyclic guanosine monophosphate (cGMP).
Objective
Several selective or non-selective inhibitors of the PDE subtypes are used clinically, i.e. sildenafil, rolipram, cysteine, etc. Recently, interest in plant-based pharmacologically bioactive compounds having potent PDEs inhibitory potential has increased. Purposely, extensive research has been carried out on natural products to explore new inhibitors of various PDEs. Therefore, this review summarizes the published data on natural PDEs inhibitors and their potential therapeutic applications.
Methods
For this purpose, natural compounds with PDE inhibitory potential have been surveyed through several databases, including PubMed, Web of Sciences (WoS), Scopus, and Google Scholar.
Results
According to a detailed literature survey, the most promising class of herbal compounds with PDE-inhibiting property has been found to belong to phenolics, including flavonoids (luteolin, kaempferol, icariin, etc.). Many other encouraging inhibitors from plants have also been identified, such as coumarins (23, 24) (licoarylcoumarin and glycocoumarin,), saponins ( agapanthussaponins), lignans (31, 33) [(±)-schizandrin and kobusin], terpenes (28, 29, 31) (perianradulcin A, quinovic acid, and ursolic acid), anthraquinones (18, 19) (emodin and chrysophanol), and alkaloids (Sanjoinine-D) (36).
Conclusion
In this review, studies have revealed the PDE-inhibitory potential of natural plant extracts and their bioactive constituents in treating various diseases; however, further clinical studies comprising synergistic use of different therapies (synthetic & natural) to acquire multi-targeted results might also be a promising option.
Cadmium has several toxic effects to human health, including carcinogenicity, nephrotoxicity, hepatotoxicity, cardiovascular, reproductive toxicities, neurotoxicity, among others. This chapter will focus mainly on cadmium toxicity: from its analytical aspects to neuronal impairment. In this context, the knowledge of analytical methods to quantify cadmium is extremely needed. The normal range of cadmium in different biological samples, and the treatment that is necessary to measure cadmium on them will be presented. In addition, the literature data about cadmium neurotoxicity bringing parameters of the cholinergic system, purinergic system, Na⁺,K⁺-ATPase activity, oxidative stress, neurotransmitter imbalance and histological brain changes will be discussed. Cd acts as a neurotoxin; a scientific fact. However, the findings underline the need for further research in the field of Cd-induced neurotoxicity, as a deeper understanding of the mechanisms underlying the neurobehavioral impairment could shed more light on the causes of its well-established cognitive implications. It is necessary to look at gender and age differences in Cd susceptibility and the role of epigenetics in determining long-term and late-onset health effects from Cd. Finally, measures should be taken to reduce Cd exposure in the general population in order to minimize the risk of health damage.
Glioblastoma (GBM) is the most frequent and aggressive brain tumor in adults and the current treatments only have a modest effect on patient survival. Recent studies show that bozepinib (BZP), a purine derivative, has potential applications in cancer treatment. The aim of this study was to evaluate the effect of BZP against GBM cells, specially concerning the purinergic system. Thus, GBM cells (C6 and U138 cell lines) were treated with BZP and cell viability, cell cycle, and annexin/PI assays, and active caspase-3 measurements were carried out. Besides, the effect of BZP over the purinergic system was also evaluated in silico and in vitro. Finally, we evaluate the action of BZP against important markers related to cancer progression, such as AKT, NF-κB, and CD133. We demonstrate here that BZP reduces GBM cell viability (IC50 = 5.7 ± 0.3 µM and 12.7 ± 1.5 µM, in C6 and U138 cells, respectively), inducing cell death through caspase-dependent apoptosis, autophagosome formation, activation of NF-κB, without any change in cell cycle progression or on the Akt pathway. Also, BZP modulates the purinergic system, inducing an increase in CD39 enzyme expression and activity, while inhibiting CD73 activity and adenosine formation, without altering CD73 enzyme expression. Curiously, one cycle of treatment resulted in enrichment of GBM cells expressing NF-κB and CD133⁺, suggesting resistant cells selection. However, after another treatment round, the resistant cells were eliminated. Altogether, BZP presented in vitro anti-glioma activity, encouraging further in vivo studies in order to better understand its mechanism of action.
Extracellular nucleotides, including adenosine triphosphate (ATP), adenosine 5′-diphosphate (ADP), uridine 5′-triphosphate (UTP) and uridine 5′-diphosphate (UDP), released under inflammation or infection conditions, are important danger-associated signaling molecules that regulate host immune responses and inflammation by binding to and activating specific surface-localized ionotropic P2X and metabotropic P2Y purinergic receptors on immune cells in an autocrine and/or paracrine manner. In this review, we summarize recent studies regarding the molecular mechanisms of immune challenge-induced ATP release in fish, the role of extracellular ATP signaling in the activation of fish innate immune responses, the purinergic receptors involved in regulating fish innate immune responses, the crosstalk between nucleotide oligomerization domain-like receptor (NLR) inflammasome signaling and extracellular ATP-mediated immune signaling, and the role of ectonucleoside triphosphate diphosphohydrolases in regulating extracellular ATP-induced innate immune responses in fish. In addition, we also discuss the immunomodulatory role of other extracellular nucleotides, including UTP, UDP and ADP, in fish.
Ectonucleotidases are key for purinergic signaling. They control the duration of activity of purinergic receptor agonists. At the same time, they produce hydrolysis products as additional ligands of purinergic receptors. Due to the considerable diversity of enzymes, purinergic receptor ligands and purinergic receptors, deciphering the impact of extracellular purinergic receptor control has become a challenge. The first group of enzymes described were the alkaline phosphatases – at the time not as nucleotide-metabolizing but as nonspecific phosphatases. Enzymes now referred to as nucleoside triphosphate diphosphohydrolases and ecto-5’-nucleotidase were the first and only nucleotide-specific ectonucleotidases identified. And they were the first group of enzymes related to purinergic signaling. Additional research brought to light a surprising number of ectoenzymes with broad substrate specificity, which can also hydrolyze nucleotides. This short overview traces the development of the field and briefly highlights important results and benefits for therapies of human diseases achieved within nearly a century of investigations.
Signal perception in plants is determinant to trigger specific physiological changes that confer resistance during the early stages of the insect attack. Thus, molecular characterization of both insect effectors and plant hormonal regulators are essential in developing forage grasses resistant to infestation by spittlebug Mahanarva spectabilis. We evaluated the presence of effectors in the salivary glands of M. spectabilis that could enable the infestation of elephant grass (Pennisetum purpureum). The presence of an NTPDase-like enzyme was confirmed, and higher activity was observed in females that hydrolyzed ADP. High levels of long chain fatty acids, such as octadecanoid acid (19.5%), prostaglandin A2 (4.7%), eicosanoid acid and the phytohormone salicylic acid (~4.0 μg/g) were also detected in the salivary glands. After 24 h of infestation, the phytohormone profiles were altered in leaves damaged by M. spectabilis, suggesting that the insect effectors modulate the plant response. Our results suggest that the plant response modulation may be the result of crosstalk between the salicylic acid, jasmonic acid, abscisic acid and zeatin pathways, induced bymolecules fromthe salivary glands. This shows that the spittlebugs can interfere with jasmonic acid and zeatin accumulation in elephant grass plants.
Trichomoniasis, one of the most common non-viral sexually transmitted infections worldwide, is caused by the parasite Trichomonas vaginalis. The pathogen colonizes the human urogenital tract and the infection is associated with complications such as adverse pregnancy outcomes, cervical cancer, and an increase in HIV transmission. The mecha-nisms of pathogenicity are multifactorial, and controlling immune responses is essential for infection maintenance. Extra-cellular purine nucleotides are released by cells in physiological and pathological conditions, and they are hydrolyzed by enzymes called ecto-nucleotidases. The cellular effects of nucleotides and nucleosides occur via binding to purinoceptors, or throughthe uptake by nucleoside transporters. Altogether, enzymes, receptors and transporters constitute the purinergic signaling, a cellular network that regulates several effects in practically all systems including mammals, helminths, proto-zoa, bacteria, and fungi. In this context, this review updates the data on purinergic signaling involved in T. vaginalis biol-ogy and interaction with host cells, focusing on the characterization of ecto-nucleotidases and on purine salvage pathways. The implications of the final products, the nucleosides adenosine and guanosine, for human neutrophil response and vagi-nal epithelial cell damage reveal the purinergic signaling as a potential new mechanism for alternative drug targets.
Distinct populations of Trypanosoma cruzi interact with mammalian cardiac muscle cells causing different inflammation patterns and low heart functionality. During T. cruzi infection, the extracellular ATP is hydrolyzed to tri- and/or diphosphate nucleotides, based on the infectivity, virulence, and regulation of the inflammatory response. T. cruzi carries out this hydrolysis through the T. cruzi ectonucleotidase, NTPDase-1 (TcNTPDase-1). This study aimed to evaluate the role of TcNTPDase-1 in culture rich in metacyclic trypomastigote forms (MT) and cell culture-derived trypomastigote forms (CT) from Colombiana (discrete typing unit - DTU I), VL-10 (DTU II), and CL (DTU VI) strains of T. cruzi. For this, we measured TcNTPDase-1 activity in suramin-treated and untreated parasites and infected J774 cells and C57BL/6 mice with suramin pre-treated parasites to assess parasitic and inflammatory cardiac profile in the acute phase of infection. Our data indicated a higher TcNTPDase-1 activity for ATP in culture rich in metacyclic trypomastigote forms from Colombiana strain in comparison to those from VL-10 and CL strains. The cell culture-derived trypomastigote forms from CL strain presented higher capacity to hydrolyze ATP than those from Colombiana and VL-10 strains. Suramin inhibited ATP hydrolysis in all studied parasite forms and strains. Suramin pre-treated parasites reduced J774 cell infection and increased nitrite production in vitro. In vivo studies showed a reduction of inflammatory infiltrate in the cardiac tissues of animals infected with cell culture-derived trypomastigote forms from suramin pre-treated Colombiana strain. In conclusion, TcNTPDase-1 activity in trypomastigotes forms drives part of the biological characteristics observed in distinct DTUs and may induce cardiac pathogenesis during T. cruzi infection.
The ubiquitous presence of aluminum in the environment leads to a high likelihood of human exposure. Neurotoxicity of the trivalent cationic form of this metal (Al3+) occurs in the central nervous system via Al-induced accumulation in cells of neural origin, including neural progenitor cells (NPCs). NPCs play a key role in the development and regeneration of the brain throughout life; therefore, this metal may contribute to neuropathological conditions. Here, we evaluated the effects of different Al³⁺ concentrations (0–50 μM) on the purinergic system and ERK pathway of NPCs isolated from embryonic telencephalons, cultured as neurospheres. Al³⁺ adhered to the cell surface of neurospheres reducing extracellular ATP release, as well as ATP, ADP, and AMP hydrolysis by NTPDase and 5′-nucleotidase, respectively. In addition, impaired nucleotide release by Al³⁺ reduced P2Y1R and A2AR expression in differentiated neurospheres. These receptors are crucial for NPC proliferation during brain development and self-repair against external stimuli, such as metal exposure. Further, Al³⁺ reduced p-ERK1/2 expression, which can diminish NPC proliferation and migration, resulting in neurogenic alterations. Thus, Al³⁺ represents an environmental agent linked to neurological disease development by alterations in ATP and ERK signal pathways, two important mechanisms associated synergistically with NPC proliferation and brain degeneration.
Ecto-nucleoside triphosphate diphosphohydrolases (ENTPDases) are pivotal regulators of extracellular ATP-mediated purinergic immune signaling. ENTPDase2 is a member of the cell surface-bound ecto-nucleoside triphosphate diphosphohydrolase (ENTPDase) protein family that hydrolyzes extracellular nucleoside 5'-triphosphates and nucleoside 5'-diphosphates. However, the immune relevance of ENTPDase2 in fish has not been elucidated. In the present study, from a comparative immunological perspective, we functionally characterized two ENTPDase2 transcript variants (namely ENTPDase2 and ENTPDase2a) from Japanese flounder (Paralichthys olivaceus). Sequence analysis indicates that the deduced Japanese flounder ENTPDase2 and ENTPDase2a proteins possess two conserved transmembrane domains and five conserved apyrase regions that are present in ENTPDase family proteins. However, these proteins only share 54% amino acid sequence identity. Tissue expression analysis revealed that both ENTPDase2 and ENTPDase2a mRNA transcripts are ubiquitously expressed in all examined Japanese flounder tissues, whereas ENTPDase2 is dominantly expressed in blood and ENTPDase2a is abundantly expressed in muscle. Immune challenge experiments showed that ENTPDase2 and ENTPDase2a were significantly upregulated by both inflammatory stimulation and Edwardsiella tarda infection. In addition, the expression of ENTPDase2 and ENTPDase2a was modulated by extracellular ATP (eATP) stimulation in a dose-dependent manner. Furthermore, immunolocalization and functional studies demonstrated that both ENTPDase2 and ENTPDase2a are functional glycosylated plasma membrane proteins. However, ENTPDase2a exhibits greater activity in the hydrolysis of eATP than ENTPDase2 and ENTPDase1 proteins. Finally, knockdown of the ENTPDase2 gene by small interfering RNA significantly upregulated the expression of eATP-induced pro-inflammatory cytokines IL-1beta, TNF-alpha and G-CSF in Japanese flounder head kidney macrophages, while knockdown of ENTPDase2a only upregulated eATP-induced IL-1beta expression. Taken together, our findings suggest that the two functional Japanese flounder ENTPDase2 isoforms play an essential role in the downregulation of eATP-induced proinflammatory cytokine expression in fish by degrading the available ATP levels in the extracellular milieu.
Extracellular adenosine triphosphate (eATP), released following inflammatory stimulation or infection, is a potent signaling molecule in activating innate immune responses in fish. However, the regulation of eATP-mediated innate immunity in fish remains unknown. Ecto-nucleoside triphosphate diphosphohydrolase 1 (CD39) is a critical molecular switch for controlling the ATP levels in the extracellular space. CD39 plays a key role in regulating eATP-activated innate immune responses through the phosphohydrolysis of pro-inflammatory eATP to inactive AMP. Here, we identified and characterized a CD39 homolog (namely, poCD39) in the Japanese flounder Paralichthys olivaceus and analyzed its regulatory role in eATP-mediated innate immunity. Real-time quantitative PCR analysis revealed that poCD39 is ubiquitously present in all tested normal tissues with dominant expression in enriched Japanese flounder head kidney macrophages (HKMs). Immune challenge experiments demonstrated that poCD39 expression was upregulated by inflammatory stimulation and Edwardsiella tarda infection. Biochemical and immunofluorescence analysis revealed that poCD39 is a functional glycosylated membrane protein for the hydrolysis of eATP. Inhibition of poCD939 activity with the ecto-NTPDase inhibitor ARL 67156 resulted in increased IL-1beta gene expression and ROS production in Japanese flounder HKMs. In contrast, overexpression of poCD39 in Japanese flounder FG-9307 cells reduced eATP-induced pro-inflammatory cytokine IL-1beta gene expression. Finally, poCD39 expression was significantly induced by eATP stimulation in the HKMs, suggesting that eATP may provide a feedback mechanism for transcriptional regulation of fish CD39. Taken together, we identified and characterized a functional fish CD39 protein involved in regulating eATP-mediated innate immune responses in fish.
A new series of raloxifene sulfonate/sulfamate derivatives were designed and synthesized. The target compounds were tested for inhibitory effect against nucleotide pyrophosphatase/phosphodiesterase-1 and -3 (NPP1 and NPP3) enzymes. Furthermore, all the ten target compounds were subjected to cytotoxic studies on various cancer cell lines, and the most potent derivatives were explored for their potency against these cancer cell lines as well as F180 fibroblasts to investigate the selectivity indexes. Compound 1f exerted the highest potency against HT-29 colon cancer cell line (IC50 = 1.4 μM) with 8.43-fold selectivity towards HT-29 than F180 fibroblasts. Compound 1f exerted sub-micromolar IC50 values against NPP1 and NPP3 (IC50 = 0.29 μM and 0.71 μM, respectively). The most potent inhibitors were docked in developed homology model of NPP1 and crystal structure of NPP3. All the docked analogues manifested remarkable interactions within the active pocket of NPP1 and NPP3.
This study aimed to evaluate nucleoside triphosphate diphosphohydrolase (NTPDase) and adenosine deaminase (ADA) activities in lymphocytes from rats supplemented or not with curcumin 30 days prior to experimental infection with Trypanosoma evansi. Thirty-two adult male Wistar rats were divided in four groups. The pre-infection group 20 (PreI20) received orally 20 mg/kg of curcumin and pre-infection group 60 (PreI60) received orally 60 mg/kg of curcumin for 30 days prior inoculation with T. evansi. The infected e non-infected control groups received only oral vehicle for 30 days. Trypanosoma evansi infected groups were inoculated intraperitoneally with 0.2 ml of blood with 1 × 106 parasites. After inoculation the treatment of the groups continued until the day of euthanasia (15 days). The results showed that curcumin pre-treatment, with both doses, reduced (P < .05) NTPDase and increased (P < .05) ADA activity in lymphocytes of treated groups when compared to untreated and infected animals (control). The results of this study support the evidence that the regulation of ATP and adenosine levels by NTPDase and ADA activities appear to be important to modulate the immune response in T. evansi infection, once the treatment with curcumin maintained the NTPDase activity reduced and enhanced ADA activity in lymphocytes. It is possible to conclude that the use of curcumin prior to infection with T. evansi induces immunomodulatory effects, favoring the response against the parasite.
Parkinson’s disease (PD) is the second most common neurodegenerative disorder, characterized by progressive loss of dopaminergic neurons that results in characteristic motor and non-motor symptoms. l-3,4 dihydroxyphenylalanine (l-DOPA) is the gold standard therapy for the treatment of PD. However, long-term use of l-DOPA leads to side effects such as dyskinesias and motor fluctuation. Since purines have neurotransmitter and co-transmitter properties, the function of the purinergic system has been thoroughly studied in the nervous system. Adenosine and adenosine 5′-triphosphate (ATP) are modulators of dopaminergic neurotransmission, neuroinflammatory processes, oxidative stress, excitotoxicity and cell death via purinergic receptor subtypes. Aberrant purinergic receptor signalling can be either the cause or the result of numerous pathological conditions, including neurodegenerative disorders. Many data confirm the involvement of purinergic signalling pathways in PD. Modulation of purinergic receptor subtypes, the activity of ectonucleotidases and ATP transporters could be beneficial in the treatment of PD. We give a brief summary of the background of purinergic signalling focusing on its roles in PD. Possible targets for pharmacological treatment are highlighted.
Two new and efficient methods for the synthesis of benzo[b]carbazolediones have been developed. Various derivatives were synthesized by either a three‐component one‐pot reaction or a two‐component domino reaction in moderate to good yield. Moreover, inhibition studies of these compounds against nucleotide pyrophosphatases (NPPs) have been carried out. An interesting and promising inhibitory effect was observed by the influence of different substituents. Substitution with a methyl group located at the indole moiety was found sensitive towards h‐NPP1 (4b; IC50 ±SEM = 0.57 ± 0.05 μM), while the unsubstituted derivative exhibited a higher sensitivity towards h‐NPP3 (4a; IC50 ± SEM = 0.16 ± 0.06 μM). Both derivatives presented non‐selective inhibition of both isozymes. Among all the derivatives, two derivatives with anisyl groups showed the highest selectivity towards h‐NPP3 and no interaction with h‐NPP1. Finally, the free binding energies were calculated and molecular docking studies were performed in order to provide an insight into putative binding modes of these inhibitors.
In humans, deficiency of the tissue non-specific alkaline phosphatase (TNAP) gene is associated with defective skeletal mineralization. In contrast, mice lacking TNAP generated by homologous recombination using embryonic stem (ES) cells have normal skeletal development. However, at approximately two weeks after birth, homozygous mutant mice develop seizures which are subsequently fatal. Defective metabolism of pyridoxal 5'-phosphate (PLP), characterized by elevated serum PLP levels, results in reduced levels of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in the brain. The mutant seizure phenotype can be rescued by the administration of pyridoxal and a semi-solid diet. Rescued animals subsequently develop defective dentition. This study reveals essential physiological functions of TNAP in the mouse.
We have previously reported the presence of a novel nucleoside triphosphate hydrolase (NTPase) in the rapidly multiplying tachyzoite form of a virulent strain (RH) of Toxoplasma gondii. On further examination, it was found that the purified enzyme was not a single enzyme but was a mixture of two isozymes termed NTPase-I and NTPase-II.
The two isozymes had similar molecular masses of approximately 240–270 kDa by gel filtration and contained four identical subunits of molecular mass 66–67 kDa by SDS-polyacrylamide gel electrophoresis. Both forms of the NTPase were activated by dithiothreitol, and NTPase-I had a specific activity 4.5-fold higher than NTPase-II in hydrolysis of ATP. The primary difference between these isozymes lies in their ability to hydrolyze nucleoside triphosphate versus diphosphate substrates. While NTPase-II hydrolyzed ATP to ADP and ADP to AMP at almost the same rate, NTPase-I hydrolyzed ADP to AMP at a much slower rate (0.7% of the rate for ATP).
The complete cDNAs for NTPase-I and NTPase-II were sequenced and found to encode the same size predicted open reading frame of which only 16 of 628 amino acids differed between the two isozymes. Both forms of the NTPase contained an NH2-terminal hydrophobic signal peptide, consistent with our previous findings that these enzymes are secreted into the host cell vacuole occupied by the parasite. The gene encoding NTPase-II was found in all strains of Toxoplasma, while the NTPase-I was confined only to virulent strains. Expression of this highly active ATPase (NTPase-I) may contribute to intracellular survival and virulence of T. gondii.
Mouse primordial germ cells express tissue non-specific alkaline phosphatase (TNAP) during development, but the widespread expression of another alkaline phosphatase gene in the early embryo limits the potential use of this marker to trace germ cells. To attempt to identify germ cells at all stages during embryonic development and to understand the role of TNAP in germ cell ontogeny, mice carrying a beta geo (lacZ/neor) disrupted allele of the TNAP gene were generated by homologous recombination in embryonic stem cells. Using beta-galactosidase activity, the embryonic pattern of TNAP expression was examined from the blastocyst stage to embryonic day 14. Results indicate that primordial germ cell progenitors do not express TNAP prior to gastrulation although at earlier times TNAP expression is found in an extraembryonic lineage destined to form the chorion. In homozygous mutants, primordial germ cells appear unaffected indicating that TNAP is not essential for their development or migration.
Current models for nucleotide sugar use in the Golgi apparatus predict a critical role for the lumenal nucleoside diphosphatase. After transfer of sugars to endogenous macromolecular acceptors, the enzyme converts nucleoside diphosphates to nucleoside monophosphates which in turn exit the Golgi lumen in a coupled antiporter reaction, allowing entry of additional nucleotide sugar from the cytosol. To test this model, we cloned the gene for the S. cerevisiae guanosine diphosphatase and constructed a null mutation. This mutation should reduce the concentrations of GDP-mannose and GMP and increase the concentration of GDP in the Golgi lumen. The alterations should in turn decrease mannosylation of proteins and lipids in this compartment. In fact, we found a partial block in O- and N-glycosylation of proteins such as chitinase and carboxypeptidase Y and underglycosylation of invertase. In addition, mannosylinositolphosphorylceramide levels were drastically reduced.
ATP diphosphohydrolase from tegumental membranes of Schistosoma mansoni was solubilized with Triton X-100 plus deoxycholate and separated by preparative nondenaturing polyacrylamide gel electrophoresis.
Two isoforms with ATP-hydrolytic activity were identified and excised from nondenaturing gels. For each of the active bands,
two protein bands (63 and 55 kDa) were detected with Coomassie Blue staining, following sodium dodecyl sulfate-polyacrylamide
gel electrophoresis. Western blots developed with polyclonal anti-potato apyrase antibody revealed a single protein of 63
kDa, either with samples excised from active bands or with total S. mansoni tegument. Anti-potato apyrase antibody immobilized on Sepharose-Protein A depleted over 95% of ATPase and ADPase activities
from detergent-solubilized tegument. Confocal laser scanning microscopy showed anti-potato apyrase antibody on the outer surface
of S. mansoni tegument. A different antibody against a fusion protein derived from recently cloned Toxoplasma gondii nucleoside triphosphate hydrolase (Bermudes, D., Peck, K. R., Afifi, M. A., Beckers, C. J. M., and Joiner, K. A. (1994) J. Biol. Chem. 269, 29252-29260) revealed the same 63-kDa band in Western blots of S. mansoni tegument. Since anti-potato apyrase antibodies exhibited cross-reactivity with S. mansoni ATP diphosphohydrolase, we decided to gain further information on the primary structure of potato apyrase by sequencing the
protein. Three novel peptides were obtained: amino-terminal sequence and two internal sequences from tryptic fragments. Eight
sequences recently deposited in the data bank, including that of T. gondii nucleoside triphosphate hydrolase, have considerable homologies to potato apyrase suggesting a new family of nucleoside triphosphatases
which contains a conserved motif (I/V)(V/M/I)(I/L/F/C)DAGS(S/T) near the amino-terminal. Antibody cross-reactivities in the
present work suggest that conserved epitopes from S. mansoni ATP diphosphohydrolase are present in this family of nucleotide-splitting enzymes.
Ecto-nucleotidases are cell surface-located enzymes which catalyze extra-cellular nucleotide hydrolysis. An extracellular hydrolysis pathway for nucleotides has been detected in essentially all tissues and also in a large variety of cell culture systems. Its general features include the following:
1.
Nucleoside 5′-triphosphates are sequentially metabolized to the nucleoside with nucleoside 5′-diphosphate and nucleoside 5′-monophosphate appearing as intermediate products. The nucleoside may then be further deaminated to inosine by adenosine deaminase (Franco et al. 1997).
2.
Not only ATP, ADP, and AMP but essentially all physiologically occurring purine and pyrimidine nucleotides are hydrolyzed.
3.
Extracellular hydrolysis of nucleotides is not inhibited by known inhibitors of intracellular ATPases such as P-type, F-type, and V-type ATPases.
4.
Nucleotide hydrolysis depends on divalent cations, generally millimolar concentrations of either Ca2+ or Mg2+.
5.
Nucleotide hydrolysis has an alkaline pH optimum.
6.
A major function of the extracellular enzyme chain appears to be the termination of the physiological action of nucleotides released from cells.
To date no information is available as to whether the hydrolysis of ATP is used to drive energy-dependent processes (for reviews of the earlier work see Arch and Newsholme 1978; Fox 1978; Pearson 1985; Dhalla and Zhao 1988; Ziganshin et al. 1994a; Plesner 1995; Sarkis et al. 1995; Beaudoin et al. 1996; Zimmermann 1996a,b; Plesner et al. 1997; Zimmermann and Pearson 1998).
Extracellular nucleotides such as ATP, or diadenosine polyphosphates and also adenosine play important receptor-mediated functions in the cardiovascular system by contributing to the regulation of vascular tone, myocardial contractility, and the function of blood cells. Both the inactivation of extracellular nucleotides and the extracellular production of adenosine arc key functional roles of ecto-nucleotidases. The final hydrolysis product adenosine may be salvaged by adjacent cells or become deaminated extracellularly to inosine by ecto-adenosine deaminase. The extracellular hydrolysis pathways have been identified by vascular perfusion or by analysis of isolated endothelial or smooth muscle cells or of cardiomyocytes. In principle, a considerable variety of enzymes can be involved in these catalytic functions. The diadenosine polyphosphatases have not yet been identified in molecular terms. Recent progress has led to the molecular cloning and functional expression of members of a new family of proteins that hydrolyze either nucleoside-5′-di- and/or triphosphates (ecto-ATP diphosphohydrolase, ecto-ATPase). The same compounds are substrates also of members of another family of ecto-nucleotidases, the phosphodiesterase/nucleotide pyrophosphatases. These enzymes have nucleotide pyrophosphatase as well as phosphodiesterase activity and can also cleave extracellular cAMP. The hydrolysis of extracellular AMP to adenosine is catalyzed by ecto-5′-nucleotidase. Finally, ecto-alkaline phosphatase is capable of releasing inorganic phosphate from a variety of organic compounds including the degradation of ATP, ADP, AMP. It should be noted that these enzymes have a broad substrate specificity hydrolyzing purine and pyrimidine nucleotides. The information concerning the distribution of the individual enzymes in the cardiovascular system and its specific cellular elements is still sparse. There is good support for a major role of ecto- ATP diphosphohydrolase in the degradation of ATP and ADP in the vascular system with an inhibitory effect on platelet activation. The expression of the individual enzymes may vary between cells in culture and their in situ location. Future studies need to unravel the cellular distribution of the enzymes by in situhybridization and immunocytological methods, analyze their functional role by intervention with specific inhibitors, reveal their regulation of expression, and evaluate the deletion of the various ecto-nucleotidase genes.
Hypophosphatasia (HPP) features defective skeletal mineralization that manifests as rickets in newborns, infants, children, and adolescents, and as osteomalacia in adults. This chapter reviews the documentation beginning in 1988, which states that HPP is caused by loss-of-function mutations in tissue nonspecific isoenzyme of alkaline phosphatase (TNSALP), the gene that encodes the TNSALP isoenzyme. However, the pathogenesis of the defective skeletal mineralization in HPP seems primarily to result from deficient TNSALP hydrolysis of inorganic pyrophosphate, an inhibitor of mineralization. It provides a brief history of the proposed function of alkaline phosphatase (ALP) in humans, and reviews the molecular and biological chemistry of human ALPs. Furthermore, it deals with HPP in detail. Based on this understanding, it discusses the insights gained from this experiment-of-nature. Furthermore, it summarizes the refinements concerning the role of TNSALP obtained from knockout mouse studies. Subsequently, it states that prenatal diagnosis of HPP is possible. During the second trimester, fetal ultrasonography or radiography has proven helpful for perinatal HPP. From the first trimester, chorionic villus samples have been used successfully for TNSALP mutation detection. However, the considerable number and variety of TNSALP mutations, as well as the influence of other factors on the HPP phenotype, makes prognostication difficult. However, there is no established medical treatment for HPP.
For the first time, a soluble, dedicated E-type ecto-ATPase has been identified and purified. This fully soluble ecto-ATPase is released into the growth media of the single-celled eukaryote,Tetrahymena,at a constant rate over time (independent of the growth phase of the cells) and it has characteristics similar to those previously described for the membrane-bound ecto-enzyme inTetrahymena.It was purified by a combination of ion-exchange, size exclusion, and affinity chromatography and nondenaturing gel electrophoresis. Its molecular weight was determined to be approximately 66,000 Da by denaturing gel electrophoresis and approximately 69,000 Da by size exclusion chromatography of the native form. The purified soluble enzyme displays the general characteristics of a dedicated E-type ecto-ATPase such as Ca2+or Mg2+dependence, hydrolysis of ATP and other nucleoside triphosphates (but not nucleoside diphosphates) and insensitivity to common ATPase inhibitors (vanadate, azide, ouabain,N-ethylmaleimide andp-chloromercuriphenyl sulfonate). It was further shown to be immunologically similar (by polyclonal antibodies) to both the membrane-bound ectoATPase of chicken gizzard smooth muscle (66 kDa) and a 66-kDa protein inTetrahymenaplasma membranes. The ecto-ATPase enzyme activity was also shown to be present in both the body plasma membrane and ciliary plasma membrane fractions but the body membrane had slightly higher specific activities. We propose that this ecto-ATPase ofTetrahymenamay play a role in inactivating purinergic signals, such as in their chemorepulsion responses to external GTP and ATP. It may also play a minor role in extracellular nucleotide scavenging.
This chapter discusses the molecular structures, catalytic properties, and functional roles of ecto-nucleotidases in the nervous system. Only one ecto-5’-nucleotidase gene is known in vertebrates. Ecto-5’-nucleotidase plays a critical role in the extracellular hydrolysis cascade of nucleotides and in neural development. Several families of ectonucleotidases function in the nervous system. They differ in their catalytic and other functional properties and consist of several members each. The chapter presents that nucleotidergic signaling pathways are widely distributed in the nervous system and that the ecto-nucleotidases play a significant part in it. The functional properties of the presently known ecto-nucleotidases and also of the sequenced but uncharacterized potential ecto-nucleotidases need to be investigated. An evaluation of their role in defined signaling pathways requires that the cellular location of the enzymes is determined. Knock-out mice in which individual ecto-nucleotidases have been deleted from the germline promise to be important tools to unravel their functional roles in the nervous system and also in other tissues.
Many, potentially all, inflammatory and immune cells express purinergic receptors of the P1 and P2 type. In recent years, availability of molecular probes and, in some cases, specific antibodies has initiated an investigation of their possible involvement in the inflammatory response. A very recent acquisition is that, besides expressing purinergic receptors, immune cells also secrete ATP, that, once in the extracellular milieu, undergoes hydrolysis by various hydrolases or kinases. Besides active secretion, ATP can also leak into the pericellular space as a consequence of cell damage. Although adenosine appears to have a preeminent anti-inflammatory activity, ATP by means of the P2X7 receptor seems to act as a proinflammatory mediator, a cytotoxic factor, or both. The combined activity of adenosine and ATP provides means for a fine modulation of the inflammatory response, depending on the amount of extracellular ATP, the rate of hydrolysis and the level of expression of P2 and P1 receptors. The P2X7 receptor appears to be involved in several processes relevant to inflammation (interleukin-1β release, cytotoxicity, formation of macrophage polykarions), thus, it may be an appealing target for pharmacologic intervention. Drug Dev. Res. 45:207–213, 1998. © 1998 Wiley-Liss, Inc.
Six P2X receptor subunits are currently known, encoded on different genes. The proteins deduced from their cDNAs have 379 to 472 amino acids; they are 36–48% identical. They are thought to have two transmembrane segments, with most of the protein forming a large extracellular loop. In-situ hybridization shows a widespread tissue distribution of the RNAs, with P2X4and P2X6being the receptors most heavily expressed in brain and P2X3found only in sensory ganglia. P2X1–P2X4subunits readily form channels when expressed in mammalian cells or oocytes; the number of subunits per channel is not known, although P2X2and P2X3can both contribute to the same channel when co-expressed. P2X5and P2X6express less readily, suggesting perhaps that they normally co-assemble with other subunits. Experiments in progress seek to determine the stoichiometry of the P2X receptor channel and the parts of the molecule involved in pore formation and ATP binding.
A variety of nucleotides and the nucleoside adenosine can act as extracellular signaling substances. Their function is terminated by extracellular degradation via surface-located enzymes. The breakdown products may be recycled. This review discusses recent developments in the cellular and molecular biology of enzymes involved in extracellular purine metabolism, including diadenosine polyphosphate hydrolase, ATP-diphosphohydrolase (apyrase), nucleotide pyrophosphatase, 5′-nucleotidase, alkaline phosphatase, NAD-glycohydrolase, and adenosine deaminase. The potential of the surface-located enzymes for ADP-ribosylation and phosphorylation of extracellular proteins is also briefly discussed. Drug Dev. Res. 39:337–352, 1996. © 1997 Wiley-Liss, Inc.
CD39, a 70- to 100-kDa molecule expressed primarily on activated lymphoid cells, was previously identified as a surface marker
of Epstein Barr virus (EBV)-transformed B cells. In this report, we show that an ecto-(Ca,Mg)-apyrase activity is present on EBV-transformed B cells, but not on B or T lymphomas. The coincidence between CD39 expression
and ecto-apyrase activity on immune cells suggests that CD39 may be an ecto-apyrase. This supposition is supported by the
observation that the amino acid sequence of CD39 is significantly homologous to those of several newly identified nucleotide
triphosphatases. Finally, we show that CD39 indeed has ecto-apyrase activity by expression in COS-7 cells.
Platelet thrombi and vascular inflammation are prominent features of discordant xenograft rejection. The purinergic nucleotides ATP and ADP, which are secreted from platelets and released by injured endothelial cells (EC), are important mediators of these reactions. Quiescent EC express the ectoenzyme ATP-diphosphohydrolase (ATPDase; an apyrase), which exerts an important thromboregulatory function by hydrolyzing both ATP and ADP. We have shown that ATPDase activity is rapidly lost from the surface of the EC following ischemia-reperfusion injury and during xenograft rejection. The aim of this study was to supplement ATPDase activity within xenografts by infusion of soluble apyrases, and thereby validate the importance of local ATPDase activity in the modulation of xenograft rejection. Lewis rats underwent heterotopic cardiac xenografting from guinea pigs and apyrase was administered intravenously (200 U/kg) as a single dose to evaluate effects on hyperacute rejection (HAR). This initial dose was followed by a continuous apyrase infusion (8.0 U/kg/hr) directly into the graft aorta in combination with systemic cobra venom factor (CVF) administration to deplete complement when delayed xenograft rejection (DXR) was studied. Functional apyrase levels in vivo were assessed by the capacity of blood samples taken at the time of surgery and rejection to inhibit platelet aggregation in vitro. Apyrase administration significantly prolonged graft survival in HAR and DXR. Functional assays showed inhibition of platelet aggregation suggesting effective systemic antiaggregatory effects of the administered apyrases, Histologic studies showed that apyrase administration abrogated local platelet aggregation and activation in HAR and DXR. Our data demonstrate that local administration of apyrase prolonged discordant xenograft survival. These observations emphasize the potential importance of purinergic mediators in platelet activation during xenograft rejection.
The metabolism of extracellular nucleotides plays an important role in nucleotide signalling mediated by P2 receptors. The nucleotide sequence encoding a putative human ecto-ATPase named CD39L1 was reported recently. However, the biological activity of this protein has not been established.
Based on the sequence of CD39L1 we isolated from mRNA from human ECV-304 cells a sequence encoding a 495 amino acid protein that is identical to CD39L1, with the exception that this sequence contains a 23 amino acid stretch in the putative extracellular loop that is missing in CD39L1. Partial sequence of a genomic DNA clone indicates that the CD39L1 gene corresponds to an alternative spliced form of the human ecto-ATPase.
Stable expression of isolated sequence in NIH-3T3 mouse fibroblasts conferred a marked nucleotide hydrolytic activity consistent with the activity of an ecto-ATPase.
The human ecto-ATPase hydrolyzed all naturally occurring nucleoside triphosphates in a Ca2+- or Mg2+-dependent manner. Nucleoside diphosphates were hydrolyzed at a rate approximately 5% of that of the corresponding triphosphates. The apparent Km and Vmax values were: 394±62 μM and 107±7 nmol Pi min−1 106 cells−1 for the hydrolysis of ATP, and 102±33 μM and 4±0.4 nmol Pi min−1 106 cells−1 for the hydrolysis of ADP, respectively.
In conclusion, we report here the cloning and functional expression of a human ecto-ATPase. The study of the biochemical properties and the regulatory mechanisms of ecto-ATPases of defined sequence will be valuable in the definition of their role in nucleotide signalling.
British Journal of Pharmacology (1999) 128, 396–402; doi:10.1038/sj.bjp.0702805
CD39, a 70- to 100-kDa molecule expressed primarily on activated lymphoid cells, was previously identified as a surface marker
of Epstein Barr virus (EBV)-transformed B cells. In this report, we show that an ecto-(Ca,Mg)-apyrase activity is present on EBV-transformed B cells, but not on B or T lymphomas. The coincidence between CD39 expression
and ecto-apyrase activity on immune cells suggests that CD39 may be an ecto-apyrase. This supposition is supported by the
observation that the amino acid sequence of CD39 is significantly homologous to those of several newly identified nucleotide
triphosphatases. Finally, we show that CD39 indeed has ecto-apyrase activity by expression in COS-7 cells.
P2-receptors (P2-R), which recognise extracellular ATP, represent significant targets for novel drug development regarding different pathophysiological conditions. In recent years, approximately fifteen ATP receptor subtypes have been cloned; seven of which belong to the P2X-R family (ligand-gated-ion-channel receptors). The remaining subtypes belong to the P2Y-R family (G-protein coupled receptors). These receptors have been classified based on their putative molecular structure, function, and the action of a subtype selective drug on the cloned receptor. A limited number of reports describe the identification of potent and selective P2X/P2Y agonists, thus extending the restricted arsenal of P2-R agonists consisting primarily of commercial compounds. Several new and subtype selective antagonists have been recently identified which open a new avenue of P2X or P2Y subtype selective antagonists for receptor studies. Current applications of P2-R agonists and antagonists include their use as insulin secretagogues, inhibitors of ADP-induced platelet aggregation, agents for hydration of lung mucous in cystic fibrosis (CF) patients, modulators of cardiac muscle contractility, and antineoplastic agents. This paper reviews selected P2-R related publications and patents issued between 1995 and 1998 for newly cloned P2-R, drug candidates, and the potential therapeutic applications of the drugs.
To elucidate the physiological role played by nucleoside triphosphate diphosphohydrolase (NTPDase; EC 3.6.1.5), adenine nucleotide analogues, modified on the purine ring, have been synthesized and tested as potential inhibitors. Resistance of ATP analogues to hydrolysis and their potency as NTPDase inhibitors were evaluated. For this purpose, a particulate fraction isolated from bovine spleen was used as the enzyme source. Among the synthesized analogues, 8-thiobutyladenosine 5‘-triphosphate (8-BuS-ATP) was found to be the most effective nonhydrolyzable competitive inhibitor, with an estimated Ki of 10 μM. This nonhydrolyzable analogue did not exert any P2X-receptor-mediated effect on endothelium-denuded blood vessels, from the guinea pig mesenteric bed. In agreement with this observation, infusion of the analogue did not cause any significant blood pressure variations of the precontracted vessel. Because in previous studies on isolated turkey erythrocytes and rat astrocytes 8-BuS-ATP was not able to trigger any P2Y1-receptor-mediated effect, it therefore appears that this NTPDase inhibitor does not interfere with purinergic receptors.
Adenine dinucleotides are a family of compounds consisting of two molecules of adenosine linked by a phosphate chain which can vary in length from 2 to 6. These naturally occurring compounds are present and stored in the CNS and are released to the synaptic cleft after stimulation. Diadenosine polyphosphates can interact with purine receptors mainly P2 and dinucleotide receptors. The second subtype of purinoceptors is specific for adenine dinucleotides, which mediate Ca2+ entry from the extracellular space in a process modulated by protein kinases A, C and protein phosphatases. This dinucleotide receptor is not sensitive to P1- and P2-purinergic antagonists. Nevertheless, diinosine pentaphosphate, Ip5I, is a potent antagonist of the dinucleotide receptor. The topographical distribution of these dinucleotides, studied by autoradiography, demonstrates a higher concentration in the cerebellum, olfactory bulb, facial nuclei and superior olive. This distribution suggests the involvement of adenine dinucleotides in motor, sensory and behavioural processes. © 1997 John Wiley & Sons, Ltd.
Studies of nucleotide receptors (P2-receptors) in cells and tissues are complicated by cleavage of phosphate groups from nucleotide agonist ligands by ecto-nucleotidases. Some P2 receptor antagonists may also inhibit ecto-nucleotidases, making these studies even more complex. In order to systematically approach this problem, we investigated structure–activity relationships of pyridoxal-5′-phosphate-6-azophenyl-2,4-disulfonate (PPADS) and 14 derivatives, many potent as antagonists at P2 receptors, as inhibitors of ecto-nucleotidases. The compounds were tested for their ability to inhibit enzymatic nucleotide breakdown by CHO cells stably transfected with plasmids containing the cDNA for rat ecto-apyrase (NTPDase1) and rat ecto-ATPase (NTPDase2). All inhibitors were tested at a concentration of 100 μM and ATP hydrolysis was quantified by HPLC. Maximal inhibition obtained for ecto-apyrase and ecto-ATPase was 60% and 35%, respectively. Most PPADS analogs were better inhibitors of ecto-apyrase than of ecto-ATPase. Compound 8, a phosphate derivative, inhibited ecto-apyrase with no inhibition evident at ecto-ATPase. Comparison of pharmacological data of PPADS analogs at P2 receptors as previously determined showed that four PPADS analogs exhibited selectivity for P2X nucleotide receptors. None of these compounds inhibited ecto-ATPase, while two inhibited the ecto-apyrase. Compound 14, a bisphosphate derivative, inhibited ecto-ATPase without inhibition of ecto-apyrase. This compound only weakly antagonized P2X1 receptors and was inactive at P2X2 and P2Y1 receptors, thus bearing some selectivity for ecto-ATPase. Compound 7, a 5-methylphosphonate derivative, a potent antagonist of P2X1 receptors, was inactive at ecto-apyrase and only weakly inhibitory at ecto-ATPase. Thus, PPADS modifications that enhance selectivity among ecto-nucleotidases and P2 receptors have been identified. Drug Dev. Res. 51:153–158, 2000. Published 2001 Wiley-Liss, Inc.
Many developmentally regulated membrane proteins of lymphocytes are ecto-enzymes, with their active sites on the external surface of the cell. These enzymes commonly have peptidase, phosphodiesterase or nucleotidase activity. Their biological roles are just beginning to be discovered. Although their expression is usually associated with particular stages of lymphoid differentiation, the same gene products are often expressed on the surface of certain non-lymphoid cell types outside the immune system, indicating that their functions cannot be unique to lymphocytes, nor can they be ubiquitous. The plasma cell membrane protein PC-1 (phosphodiesterase I; EC 3.1.4.1/nucleotide pyrophosphatase; EC 3.6.1.9), which was one of the first serological markers for lymphocyte subsets to be discovered, is a typical example. Within the immune system, PC-1 is confined to plasma cells, which represent about 0.1% of lymphocytes. However, PC-1 is also expressed on cells of the distal convoluted tubule of the kidney, chondrocytes, osteoblasts, epididymis and hepatocytes. Recent work has shown that PC-1 is a member of a multigene family of ecto-phosphodiesterases that currently has two other members, PD-1α (autotaxin) and PD-1β (B10). Within this family, the extracellular domains are highly conserved, especially around the active site. In contrast, the transmembrane and cytopiasmic domains are highly divergent. Individual members of the ecto-phosphodiesterase family have distinct patterns of distribution in different cell types, and even within the same cell. For example, PC-1 is present only on the basolateral surface of hepatocytes, while B10 (PD-1β) is confined to the apical surface. Analysis of conservation and differences in the sequence of their cytoplasmic tails may illuminate intracellular tar-getting signals. Ecto-phosphodiesterases may play a part in diverse activities in different tissues, including recycling of nucleotides. They may also regulate the concentration of pharmacologically active extracellular compounds such as adenosine or its derivatives and ceil motility. Some members may modulate local concentrations of pyrophosphate, and hence influence calcification in bone and cartilage.
The effect of ecto-nucleotide pyrophosphatase (ecto-NPPase; EC 3.6.1.9) on the ATP- and ADP-mediated receptor activation was studied in rat C6 glioma cells. The P2-purinoceptor antagonists pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) and reactive blue (RB2) are potent inhibitors (IC50=12±3 μM) of the latter enzyme. 4,4′-diisothiocyanatostilbene-2,2′ disulfonic acid (DIDS), 5′-phosphoadenosine 3′-phosphate (PAP) and suramin were less potent inhibitors with an IC50 of 22±4, 36±7 and 72±11 μM respectively.
P1-purinoceptor antagonists CGS 15943, cyclo-pentyl theophylline (CTP) and theophylline did not affect the activity of the ecto-NPPase.
ATP- and ADP-mediated P2Y1-like receptor activation inhibited the (−)-isoproterenol-induced increase of intracellular cyclic AMP concentration. PPADS, an ineffective P2Y-antagonist in C6, potentiated the ATP and ADP effect approximately 3 fold due to inhibition of nucleotide hydrolysis by the ecto-NPPase.
We conclude that ecto-NPPase has a modulatory effect on purinoceptor-mediated signalling in C6 glioma cell cultures.
British Journal of Pharmacology (2000) 130, 139–145; doi:10.1038/sj.bjp.0703289
Extracellular ATP can produce various effects acting via P2-purinoceptors. ATP is rapidly broken down by ecto-ATPase and other ecto-enzymes that limit its effect. Further, adenosine, a metabolite of ATP breakdown, can produce its own effect acting via P1-purinoceptors, sometimes masking the effects of ATP. An inhibitor of ATP degradation would be a useful pharmacological tool to discriminate between effects of ATP and its metabolites, as well as to potentiate its actions. Diverse compounds that have been claimed to be inhibitors of ATP-metabolising ectoenzymes are evaluated, but specific and selective Ca2+ /Mg2+ -dependent ecto-ATPase inhibitors still appear to be lacking.
The role of ecto-ATPase in modulating the purinergic component of neurotransmission in the guinea-pig vas deferens has been investigated using ARL 67156, a recently developed inhibitor of ecto-ATPase. ARL 67156 rapidly and reversibly potentiated neurogenic contractions in a concentration-dependent manner. ARL 67156 also potentiated contractions evoked by exogenous ATP, but had no effect on those to the stable analogue α,β-methyleneATP or on those to noradrenaline and KCl in the presence of the P2-purinoceptor antagonist PPADS. These results are consistent with an inhibitory action of ARL 67156 on ecto-ATPase and suggest that ecto-ATPase modulates purinergic neurotransmission in the guinea-pig vas deferens.
CD73 or ecto-5′-nucleotidase (S-NT) is a widely expressed ecto-enzyme which catalyzes the dephosphorylation of AMP and other nucleoside monophosphates. CD 73 participates in purine salvage through this enzymatic activity, supplying cells with precursors for energy metabolism and nucleic acid biosynthesis. As an enzyme that produces adenosine, CD 73 can also regulate adenosine receptor engagement in many tissues. However, CD73 also has functions independent of its enzyme activity like many glycosyl phosphatidylinositol (GPI)-anchored molecules, it transmits potent activation signals in T cells when ligated by antibodies. Less compelling evidence suggests that CD 73 may function as a cell adhesion molecule. In the human immune system, CD73 is expressed on subsets of T and B cells, on germinal center follicular dendritic cells, and on thymic medullary reticular fibroblasts and epithelial cells. Many challenging areas remain to be explored before the role of CD73 in the immune system will be fully understood. These include an evaluation of the role of adenosine receptors in lymphoid development, the identification of physiological CD73 ligands, a functional assessment of the GPI anchor, and an analysis of the intricate cell-type-specific and developmental regulation of CD 73 expression.
Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) comprise a novel family of ectonucleotidases that are important in the hydrolysis of extracellular nucleotides. The related NTPDase1 (ecto-apyrase) and NTPDase2 (ecto-ATPase) share a common membrane topography with a transmembrane domain at both the N- and C-terminus, an extensive extracellular loop with five ‘apyrase conserved regions’ (ACR1 to ACR5), and a cysteine-rich C-terminal region. Whereas NTPDase1 expressed in CHO cells hydrolyzes ATP and ADP equivalently, NTPDase2 has a high preference for the hydrolysis of ATP over ADP. In addition recombinant NTPDase1 hydrolyzes ATP to AMP with the formation of only minor amounts of free ADP. In contrast, ADP appears as the major free product when ATP is hydrolyzed by NTPDase2. In order to determine molecular domains responsible for these differences in catalytic properties, chimeric cDNAs were constructed in which N-terminal sequences of increasing length of NTPDase1 were substituted by the corresponding sequences of NTPDase2 and vice versa. The turnover points were contained within ACR1 to ACR5. Chimeric cDNAs were expressed in CHO cells and surface expression was verified by immunocytochemistry. ATP and ADP hydrolysis rates and ADP and AMP product formation were determined using HPLC. Amino-acid residues between ACR3 and ACR5 and in particular the cysteine-rich region between ACR4 and ACR5 conferred a phenotype to the chimeric enzymes that corresponded to the respective wild-type enzyme. Protein structure rather than the conserved ACRs may be of major relevance for determining differences in the catalytic properties between the related wild-type enzymes.
Stimulation of endothelial cells from human umbilical vein by shear stress induced release of endogenous ATP which was accompanied by an extracellular increase in the activity of enzymes degrading both ATP (ATPases) and AMP (5′-nucleotidases).
The activity of soluble ATPase was progressively increased from 1.62±0.27 to 12.7±1.0 pmoles ml−1 h−1 after 60 min of stimulation by shear stress.
The rate of [3H]-ATP hydrolysis in the medium was inhibited by the purinergic agents suramin, Reactive blue 2 and pyridoxalphosphate-6-azophenyl-2′4′-disulphonic acid, and remained insensitive to the classic inhibitors of ion-pumping and intracellular ATPases.
Shear stress also increased the activity of 5′-nucleotidase in the medium from 2.0±0.5 to 27.2±2.8 pmoles ml−1 h−1. When shear stress was applied after removal of ecto-5′-nucleotidase by phosphatidylinositol-specific phospholipase C, the release of 5′-nucleotidase was drastically reduced.
These results show that soluble ATPase and 5′-nucleotidase which are released during shear stress are not released from an intracellular compartment together with ATP but have an extracellular origin.
British Journal of Pharmacology (2000) 129, 921–926; doi:10.1038/sj.bjp.0703136
We report the inactivation, via homologous recombination, of two of the three active mouse alkaline phosphatase genes, i.e., embryonic (EAP) and tissue nonspecific (TNAP). Whereas expression of the EAP isozyme was abolished in all tissues that express EAP developmentally (such as the preimplantation embryo, thymus, and testis), the EAP knock-out mice show no obvious phenotypic abnormalities. They reproduce normally and give birth to live offspring, indicating the nonessential role of EAP during embryonic development. Mice deficient in the TNAP gene mimic a severe form of hypophosphatasia. These TNAP −/− mice are growth impaired, develop epileptic seizures and apnea, and die before weaning. Examination of the tissues indicates abnormal bone mineralization and morphological changes in the osteoblasts, aberrant development of the lumbar nerve roots, disturbances in intestinal physiology, increased apoptosis in the thymus, and abnormal spleens. Our results indicate that, in the mouse, TNAP appears not to be essential for the initial events leading to bone mineral deposition but that TNAP seems to play a role in the maintenance of this process after birth. The other phenotypic manifestations may be a consequence of the lack of TNAP in the developing neural tube between stages E8.5 and E13.5 of embryogenesis. We hypothesize that the autonomic nervous system is compromised in these TNAP −/− mice. Dev. Dyn. 208:432–446, 1997. © 1997 Wiley-Liss, Inc.
Extracellular nucleotides are ubiquitous extracellular mediators that interact with and activate nucleotide type 2 (P2) receptors. These receptors initiate a wide variety of signalling pathways that appear important for functional associations between neurons and glial cells and for the regulation of blood flow, haemostatic and inflammatory reactions in the brain. Ectonucleotidases are extracellular nucleotide-metabolizing enzymes that modulate P2 receptor-mediated signalling by the regulated hydrolysis of these agonists. A considerable number of ectoenzyme species with partially overlapping substrate and tissue distributions have been described. Major candidates for expression in the brain are members of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase or CD39) family. The production of cd39–/–mice and specific reagents have enabled us to analyse the specific cellular distribution of NTPDase1 (CD39), the prototype member of the enzyme family, in the mouse brain. Using monospecific antibodies and enzyme histochemical staining, we have identified NTPDase1 as a major ectonucleotidase associated with both microglia and the endothelial and smooth muscle cells of the vasculature. NTPDase1 is not expressed by neurons and astrocytes. Additional unidentified ectonucleotidase functional activity is observed at lower levels throughout the brain parenchyma. NTPDase1 may regulate P2 receptor-mediated functions of microglia as well as influence nucleotide signalling between neurons or astrocytes that are associated with multiple microglial ramifications. The expression of NTPDase1 by cerebrovascular endothelial and smooth muscle cells also suggests involvement in the regulation of blood flow and thrombogenesis.
In addition to their well-established roles as neurotransmitters and neuromodulators, growing evidence suggests that nucleotides and nucleosides might also act as trophic factors in both the central and peripheral nervous systems. Specific extracellular receptor subtypes for these compounds are expressed on neurons, glial and endothelial cells, where they mediate strikingly different effects. These range from induction of cell differentiation and apoptosis, mitogenesis and morphogenetic changes, to stimulation of synthesis or release, or both, of cytokines and neurotrophic factors, both under physiological and pathological conditions. Nucleotides and nucleosides might be involved in the regulation of development and plasticity of the nervous system, and in the pathophysiology of neurodegenerative disorders. Receptors for nucleotides and nucleosides could represent a novel target for the development of therapeutic strategies to treat incurable diseases of the nervous system, including trauma- and ischemia-associated neurodegeneration, demyelinating and aging-associated cognitive disorders.Trends Neurosci. (1996) 19, 13–18
CD73 (ecto-5′-nucleotidase), a glycosyl phosphatidylinositol (GPI) anchored purine salvage enzyme expressed on the surface of human T and B lymphocytes, catalyzes the conversion of purine and pyrimidine ribo- and deoxyribonucleoside monophosphates to the corresponding nucleosides. The cellular distribution, cDNA sequence, and structure of CD73 are reviewed. CD73 serves as a costimulatory molecule in activating T cells. A Jurkat T cell line transfected with the CD73 cDNA revealed that neither enzymatic activity nor the GPI anchor is necessary for T cell activation in vitro via CD73, while expression of p56lck, CD45 and the T cell receptor are required. Models for the transmission of signals via CD73 and other GPI-anchored proteins are discussed. CD73 generated adenosine functions in cell signalling in many physiologic systems, including intestinal epithelium, ischemic myocardium, and cholinergic synapses. The hypothesis that CD73 produces adenosine that is important for T cell development is presented.
Ectonucleotidases provide the signal termination mechanism for purinergic transmission, including fast excitatory neurotransmission by ATP in the CNS. This study provides evidence for ectonucleotidase expression in the rat cochlea, brain and other tissues. In addition to detection of rat ecto-ATPase and ecto-ATPDase in these tissues, we identify a novel ecto-ATPase splice variant arising from the loss of a putative exon (193 bp) in the C-terminal coding region. This is the first evidence of alternative splicing in the ecto-ATPase gene family. Splicing of the 193-bp putative exon containing a stop codon extends the open reading frame and provides translation of an additional 50 amino acids compared with the isoform isolated earlier from the rat brain (rEATPaseA; GenBank accession #Y11835). The splice variant (rEATPaseB; GenBank accession #AF129103) encodes 545 amino acids with a predicted protein molecular mass of 60 kDa. rEATPaseB contains a long cytoplasmic tail (62 amino acids) with three potential protein kinase CK2 phosphorylation sites not present in rEATPaseA. Co-expression of two ecto-ATPase isoforms with different regulatory sites suggests that the extracellular ATP signal levels may be differently influenced by intracellular feedback pathways.
Ecto-5'-nucleotidase is regarded as being the key enzyme in the formation of the neuromodulator adenosine from released ATP. However, the association of ecto-5'-nucleotidase with nerve terminals is not consensual. Only enzyme histochemical and biochemical studies, but not immunocytochemical studies, agree on a general synaptic location of the enzyme. To clarify this issue further we tested the effect of an antibody against ecto-5'-nucleotidase, previously used in immunocytochemical studies, on the activity of ecto-5'-nucleotidase in fractions of nerve terminals isolated from different areas of rat hippocampus. The specific activity of extracellular AMP catabolism was higher in synaptosomes from the CA3 area (0.81+/-0.06 nmol/min/mg of protein) than from synaptosomes from the CA1 area or the dentate gyrus or from the whole hippocampus (0.49-0.68 nmol/ min/mg of protein). The catabolism of AMP (10 microM) was equally inhibited (85-92%) in synaptosomes from whole hippocampus, CA1, CA3, or dentate gyrus by alpha,beta-methylene-ADP (100 microM) and equally unaffected by p-nitrophenyl phosphate (0.5 mM) or rabbit IgGs (100 microg/ml). However, the antiserum against ecto-5'-nucleotidase (100 microg/ml) inhibited extracellular AMP catabolism by 44% in CA3 synaptosomes but had little or no effect in synaptosomes from CA1, dentate gyrus, or whole hippocampus. A similar difference in the inhibitory potential of the antibody was observed between fractions of isolated 5'-nucleotidase binding to concanavalin A-Sepharose (70%) and fractions not retained by the lectin column (18%). Taken together, these results suggest that immunological isoforms of ecto-5'-nucleotidase exist in the rat hippocampal nerve terminals, with predominance in the CA3 area.
A variety of distinct protein glycosylation reactions occur in the endoplasmic reticulum (ER) of eukaryotic cells. In some instances, both the proteins to be glycosylated and the precursor sugar donors must be translocated across the membrane from the cytoplasm to the lumen of the ER. Elucidation of the individual steps in each of the glycosylation pathways has revealed the topographic complexity of these reactions.
5'-Nucleotidase activity in vertebrate tissues reflects the activity of four different forms of enzymes, a surface-located GPI-anchored ecto-form (e-N), a soluble form derived from it by cleavage of the membrane anchor (e-N(s)), and two cytosolic enzymes (c-N-I and c-N-II). Only the surface-located ecto-5'nucleotidase (e-N) has been characterized in molecular terms. It is expected that future sequence work will reveal that the closely related cytoplasmic forms c-N-I and c-N-II represent different genomic products. As yet the possibility of tissue-specific forms of 5'-nucleotidase as well as of splicing variants cannot be excluded. The e-N form reveals significant sequence identities with multispecific nucleotidases in bacteria, indicating common origin. Multispecific 5'-nucleotidases also occur in yeast but plant enzymes generally display marked substrate specificity. A characterization of the three-dimensional structure and of the active site would provide a basis for the interpretation of substrate specificities. The production of nucleosides is the general result of 5'nucleotidase activity. But the physiological implication of this catalytic process at thc cellular or tissue level varies. The intracellular enzymes c-N-I and c-N-II are involved in the catabolism of AMP and IMP and also of other mononucleotides and correspondingly produce the nucleoside. They are thought to be of particular importance for the hydrolysis of AMP and IMP in situations of low energy charge. The stimulatory effect of ATP and/or ADP on both affinity and rate seems to be of major physiological significance. A detailed analysis of the general biochemical properties with highly purified enzyme preparations is essential for setting the functional interpretations on a reliable basis. Furthermore, any general functional interpretation of the cytosolic enzymes requires both intimate knowledge of other cytosolic enzymes capable of hydrolysing 5'-mononucleotides as well as of the actual free concentrations of the metabolites concerned. Thc surface-located enzyme is involved in a number of functions which presumably do not mutually exclude each other. They rather supplement each other in their contribution to the physiology of cellular metabolism and tissue function. It is important to note that expression of ecto-5'-nucleotidase does not occur on all cells and can be developmentally regulated. Ecto-5'-nucleotidase is involved in the salvage of extracellular nucleotides. In addition, the enzyme has a major role in the control of tissue homeostasis. It effects the final step in the complete hydrolysis of thc extracellular messenger ATP. and, at the same time, produces adenosine as a further extracellular messenger. But this polysialyated GPI-anchored metalloprotein might not only function as an enzyme. The increased expression of the protein at the surface of activated cells and during certain stages of cell maturation, its capability to bind fibronectin as well as the presence of the HNK-I epitope in some tissues implies an involvement in cellular interactions such as cell recognition and cell adhesion or in cell matrix relations. This calls for a detailed analysis also of the carbohydrate chains, including possible developmental changes in the glycosylation pattern. Studies with lymphocytes even imply a function in signal transfer from the cell surface to the interior. Possibly a new mechanism for activation of intracellular messengers via GPI-anchored proteins will evolve. Finally, consensus should be sought on 5'-nucleotidase nomenclature. Since conventional enzyme nomenclature groups all forms described into one category the literature has become confusing. For example, at the bacterial level, periplasmic 5'nuclcotidase is multispecific and its relation to the vertebrate ecto-5'-nucleotidase can only be deduced from the primary structure. The cytolic 5'-nucleotidases have kinetic properties and physiological functions completely different from those of the ectoenzyme and the soluble form derived from it. It is suggested that current enzyme nomenclature is extended by introducing subclasses of EC 3.1.3.5: 1 for c-N-I, 2 for c-N-II, and 3 for e-N and e-N(s). 5'-Nucleotidases from bacteria and plants could then be grouped according to their sequence identities with one of the vertebrate enzymes.
The distribution, biochemical properties, and function of CD39 were characterized with the use of a new mAb termed 400. CD39 is an acidic (isoelectric point, approximately 4.2) glycoprotein of Mr approximately 78,000, containing approximately 24 kDa of N-linked oligosaccharide but no detectable O-linked sugars. CD39 was not expressed by resting blood T, B, or NK cells, neutrophils, or monocytes, but was expressed on activated NK cells, B cells, subsets of T cells, and T cell clones. Furthermore, the pattern of expression of CD39 was distinct from the "classic" activation Ag CD25 and CD71, inasmuch as it was expressed long after expression of CD25 and CD71 had returned to basal levels. CD39 was easily detectable on EBV-transformed B cell lines but was absent from pre-B and non-EBV-transformed B cell lines, most myeloid cell lines, and leukemic T cell lines. In lymphoid tissues, germinal center cells expressed little or no CD39, whereas some paracortical lymphocytes and most macrophages and dendritic cells were positive. CD39 was strongly expressed by endothelium in all tissues examined, including skin, and was present on some, but not all, endothelial cell lines propagated in vitro. Interestingly, mAb binding to certain epitopes on CD39 induced rapid homotypic adhesion that appeared to involve LFA-1 (CD11a/CD18), but was morphologically and kinetically distinct from that induced by PMA. Anti-CD39 mAb also induced homotypic adhesion in an CD11/CD18-EBV-transformed B cell line derived from a patient with severe leukocyte adhesion deficiency. This adhesion was unaffected by EDTA, suggesting that this pathway of anti-CD39-induced homotypic adhesion was not mediated by any of the known integrins. These studies suggest that CD39 is involved in the cellular signaling that regulates adhesion.
Adenylate kinase activity (ATP:AMP-phosphotransferase; EC 2.7.4.3) was studied in various subcellular fractions of rat brain tissues. Because of the presence of other adenosine nucleotide-utilizing enzymes, adenylate kinase activity was assayed in both the forward and reverse directions by using coupled enzyme systems and by using a specific adenylate kinase inhibitor, P1,P5-di(adenosine-5') pentaphosphate. As expected, the highest specific adenylate kinase activity (2.89 mumol/min/mg of protein) was detected in the cytosolic brain fraction. However, substantial enzyme activity (0.68 mumol/min/mg) was also found in the intact synaptosomal fraction isolated on Percoll/sucrose gradients. The increased specific enzyme activity of purified synaptosomes and the differences found between the kinetic parameters of the membrane-bound and cytosolic enzyme forms suggest that the synaptosomal adenylate kinase activity cannot be attributed to the small amount of contaminating cytosol present in our preparations. The adenylate kinase enzyme adhered to purified synaptic plasma membranes and was not released by washings with isoosmotic sucrose medium. The facts that the adenylate kinase enzyme activity could be measured in intact synaptosomal preparations and that both its substrates and its inhibitors do not cross intact plasma membranes support the possibility that the synaptosomal adenylate kinase is an ecto-enzyme.
Using a hippocampal subcellular fraction enriched in mossy fiber synaptosomes, evidence was obtained indicating that adenosine derived from a presynaptic pool of ATP may modulate the release of prodynorphin-derived peptides. and glutamic acid from mossy fiber terminals. Synaptosomal ATP was released in a Ca2+-dependent manner by K+-induced depolarization. The rapid hydrolysis of extracellular [14C]ATP in the presence of intact mossy fiber synaptosomes resulted in the production of [14C]adenosine. Micromolar concentrations of a stable adenosine analogue, 2-chloroadenosine, inhibited the K+-stimulated release of both dynorphin B and dynorphin A(1-8). 2-Chloroadenosine failed to suppress the evoked release of glutamic acid, measured in these same superfusates, unless the mossy fiber synaptosomes were pretreated with D-aspartic acid to deplete the cytosolic, Ca2+-independent, pool of this acidic amino acid. In synaptosomes pretreated in this manner, release of the remaining Ca2+-dependent pool of glutamic acid was significantly inhibited by NiCl2, 2-chloroadenosine, 5'-N-ethylcarboxamidoadenosine, cyclohexyladenosine, and R(-)-N6(2-phenylisopropyl)adenosine, but not by ATP. 2-Chloroadenosine-induced inhibition was reversed when the external CaCl2 concentration was raised from 1.8 mM to 6 mM. 8-Phenyltheophylline, an adenosine receptor antagonist, effectively blocked the inhibitory effects of 2-chloroadenosine on mossy fiber synaptosomes and significantly enhanced the K+-evoked release of both glutamic acid and dynorphin A(1-8) when added alone to the superfusion medium. These results support the proposition that depolarized hippocampal mossy fiber synaptosomes release endogenous ATP and are capable of forming adenosine from extracellular ATP, and that endogenous adenosine may act at a presynaptic site to inhibit the further release of glutamic acid and the prodynorphin-derived peptides.
Ecto-ATPases are ubiquitous in eukaryotic cells. They hydrolyze extracellular nucleoside tri- and/or diphosphates, and, when isolated, they exhibit E-type ATPase activity, (that is, the activity is dependent on Ca2+ or Mg2+, and it is insensitive to specific inhibitors of P-type, F-type, and V-type ATPases; in addition, several nucleotide tri- and/or diphosphates are hydrolysed, but nucleoside monophosphates and nonnucleoside phosphates are not substrates). Ecto-ATPases are glycoproteins; they do not form a phosphorylated intermediate during the catalytic cycle; they seem to have an extremely high turnover number; and they present specific experimental problems during solubilization and purification. The T-tubule Mg2+-ATPase belongs to this group of enzymes, which may serve at least two major roles: they terminate ATP/ADP-induced signal transduction and participate in adenosine recycling. Several other functions have been discussed and identity to certain cell adhesion molecules and the bile acid transport protein was suggested on the basis of cDNA clone isolation and immunological work.
Alloantigen-specific and OVA-specific CD8+ CTL were shown here to express an ectoATPase. These CTL also express an ectoADPase, but do not express detectable levels of an ectoAMPase. CD8+ CTL transported adenosine into their cytoplasm at a rate of 2.3 x 10(-11) mmol/min/10(5) cells. In contrast, adenosine uptake was 34-fold lower when ATP was used as the source of the nucleoside. This was consistent with the lack of ectoAMPase and suggests that the role of ectoATPase is not in the salvage of extracellular nucleotides. 5'-p-(fluorosulfonyl)benzoyl adenosine (5'-FSBA) is an ATP analogue affinity label that irreversibly inhibits CTL ectoATPase. Cells made ectoATPase activity deficient by modification with 5'-FSBA were not susceptible to potential lytic effects of extracellular ATP with less than 20% specific lysis at 20 mM of exogenous ATP. However, cells modified by 5'-FSBA were unable to kill their respective target cells. Complete inhibition of cell-mediated killing was observed with 1 mM 5'-FSBA. CTL modified by 5'-FSBA also failed to secrete TNF-alpha and IFN-gamma after activation by the appropriate Ag. Killing was also inhibited by 5'-adenylylimidodiphosphate (a nonhydrolyzable ATP analogue), but not by ATP, ADP, alpha, beta-methylene ADP (a nonhydrolyzable ADP analogue), AMP, or adenosine. Blockage of CTL activity by 5'-FSBA was not reversed by addition of ADP, suggesting that hydrolysis of ATP is an essential ectoATPase-mediated signal for CTL activation. These results suggest that ectoATPase is essential for Ag recognition and/or effector activities of CTL.
By taking advantage of the specific neuronal and connective organization of the hippocampus and the different susceptibility of hippocampal neurons to transient cerebral ischemia or intraventricular injections of kainic acid (KA), we examined the microglial reactions to different types of neuronal injury. In all areas with neuronal or axonal degeneration, the microglial cells reacted by specific degeneration-related morphological transformations and expression of class I major histocompatibility complex (MHC) antigen. Subpopulations of microglial cells also expressed class II MHC antigen and leukocyte common antigen (LCA) in relation to (1) degenerating nerve cell bodies in the dentate hilus and the CA1 and CA3 pyramidal cell layers, (2) postischemic degeneration of dendrites in the stratum radiatum of CA1, and (3) combined dendritic and axonal degeneration in the stratum radiatum of the KA-lesioned CA3. MHC II and LCA expression was not observed in relation to degeneration of the CA3-derived Schaffer collaterals in CA1 after KA-induced CA3 lesions. In the case of ischemia the degeneration-related reactions were preceded by an early, generalized microglial reaction, which also included areas without subsequent signs of neural degeneration. This reaction, which was transient and characterized by subtle morphological changes and induction of class I MHC antigen only, was presumably triggered by a general postischemic perturbation of the cerebral microenvironment, and not by actual neural degeneration. In conclusion, we found that microglial expression of class I MHC antigen was a sensitive marker of both the general perturbation after ischemia and axonal degeneration distant from the areas of actual nerve cell death.(ABSTRACT TRUNCATED AT 250 WORDS)
We have investigated responses to extracellular ATP (ATPe) in the microglial cell lines N9 and N13 and in freshly isolated mouse microglial cells. Upon stimulation with this nucleotide, N9 and N13 cells underwent an increase in the cytoplasmic free Ca2+ concentration ([Ca2+]i), a sustained depolarization of the plasma membrane, and an uptake of extracellular markers such as ethidium bromide and lucifer yellow; increases in plasma membrane permeability were paralleled by striking morphologic changes. ATPe, as well as other nucleotides, activated a spiking Ca2+ release from intracellular stores; however, only ATPe was also able to cause a massive transmembrane Ca2+ influx. The ATP analogue 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) triggered a sustained Ca2+ influx accompanied by little release from stores. The ATP derivative oxidized ATP (oATP) strongly inhibited Ca2+ influx, minimally affecting Ca2+ release. From ATPe-sensitive microglial cell lines, we selected several ATPe-resistant clones that showed complete lack of ATPe-mediated plasma membrane permeability changes, although they retained the Ca2+ mobilization response from intracellular stores. ATPe-dependent plasma membrane permeability changes were also greatly reduced in growth-arrested microglial cells. Finally, ATPe triggered IL-1 beta release from wild-type but not ATPe-resistant microglial cells. These results show that microglial cells express at least two purinergic receptor subtypes, metabotropic (P2Y) and ionotropic (P2Z), and that the latter is modulated during cell cycle and coupled to IL-1 beta release.
A soluble ATP-diphosphohydrolase (apyrase, EC 3.6.1.5) has been purified from potato tubers. Solanum tuberosum, to a specific activity of 10,000 mumol P(i)/mg/min. The cDNA corresponding to the potato apyrase has been isolated and termed RROP1. The deduced amino acid sequence contains a putative signal sequence, two hydrophobic regions at the carboxy terminus, two potential Asn-linked glycosylation sites, and four regions in the amino-terminal half that we term ACR (apyrase conserved regions) 1-4 that are highly conserved in known apyrases and related enzymes; garden pea nucleoside triphosphatase, Toxoplasma gondii nucleoside triphosphate hydrolases, and Saccharomyces cerevisiae golgi guanosine diphosphatase. A yeast 71.9-kDa hypothetical protein on chromosome V, a Caenorhabditis elegans hypothetical 61.3-kDa protein on chromosome III, and human CD39, a lymphoid cell activation antigen, also share the conserved ACR regions, but their ability to hydrolyze nucleotides has not been assessed.
The extracellular ATPase (ecto-ATPase) is a divalent cation-dependent nucleoside triphosphatase with an unusually high specific activity. Monoclonal antibodies, described previously [Stout, J. G., Strobel, R. S., & Kirley, T. L. (1995) J. Biol. Chem. 270, 11845-11850], and newly generated polyclonal antibodies, both raised against the chicken gizzard ecto-ATPase, were evaluated for their ability to cross-react with mammalian ecto-ATPases and were used as specific immunochemical probes to identify non-cross-linked and cross-linked ecto-ATPase. Unlike previous results obtained with the rabbit skeletal muscle ecto-ATPase enzyme, cross-linking the chicken gizzard smooth muscle ecto-ATPase with 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP) and dithiobis(succinimidylpropionate) (DSP) increased the activity of the enzyme which corresponded to an increase in a approximately 130 kDa immunoreactive band, proposed to be a ecto-ATPase homodimer, and a concomitant decrease in a approximately 66 kDa immunoreactive band, the ecto-ATPase monomer. Ecto-ATPase was immunochemically identified in chicken, rat, mouse, rabbit, and pig. Interestingly, under nonreducing conditions, the ecto-ATPase activity in rat and pig (unlike chicken and rabbit) was evident on Western blots as an immunoreactive band at approximately 200 kDa, proposed to be an intermolecularly disulfide-linked ecto-ATPase homotrimer. Nonreducing Western blot analysis of various rat tissues with three different monoclonal antibodies that recognize the 66 kDa chicken gizzard ecto-ATPase monomer strengthened the hypothesis that this 200 kDa band indeed represents the trimeric ecto-ATPase. After reduction, ecto-ATPase monomers were found to be approximately 66 kDa in all species examined. The differences in ecto-ATPase quaternary structure stability may account for the observed species differences in ecto-ATPase enzymatic properties. Intermolecular disulfide bonds appear to be one of the species-specific ways to stabilize the native, active ecto-ATPase quaternary structure (the homotrimer). Based on the data obtained, as well as previous data from this and other laboratories, a hypothesis was developed to explain the modulation of ecto-ATPase activity by a variety of agents, including detergents, chemical cross-linkers, lectins, antibodies, and small molecule inhibitors. It is proposed that agents and conditions stabilizing ecto-ATPase oligomers stimulate enzyme activity, whereas agents and conditions destabilizing ecto-ATPase homooligomers would inhibit the ecto-ATPase.
Nucleotides such as ATP, ADP, UTP or the diadenosine polyphosphates and possibly even NAD+ are extracellular signaling substances in the brain and in other tissues. Enzymes located on the cell surface catalyze the hydrolysis of these compounds and thus limit their spatio-temporal activity. As a final hydrolysis product they generate the nucleoside and phosphate. The paper discusses the biochemical properties, cellular localization and functional properties of surface-located enzymes that hydrolyse nucleotides released from nervous tissue. This is preceded by a brief discussion of nucleotide receptors, cellular storage and mechanisms of nucleotide release. In nervous tissue nucleoside 5'-triphosphates are hydrolysed by ecto-ATP-diphosphohydrolase and possibly in addition also by ecto-nucleoside triphosphatase and ecto-nucleoside diphosphatase. The molecular identity of the ATP-diphosphohydrolase has now been revealed. The hydrolysis of nucleoside 5'-monophosphates is catalysed by 5'-nucleotidase whose biochemical properties and molecular structure have been studied in detail. Little is known about the molecular properties of the diadenosine polyphosphatases. Surface located enzymes for the extracellular hydrolysis of NAD+ and also ecto-protein kinases are discussed briefly. The cellular localization of the ecto-nucleotidases is only partly defined. Whereas in adult mammalian brain activity for hydrolysis of ATP and ADP may be associated with nerve cells or glial cells 5'-nucleotidase appears to have a preferential glial allocation in the adult mammal. The extracellular hydrolysis of the nucleotides is of functional importance not only during synaptic transmission where it functions in signal elimination. It plays a crucial role also for the survival and differentiation of neural cells in vitro and presumably during neuronal development in vivo.