No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
CD39 is an ecto-(Ca, Mg)-apyrase
Abstract
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.
... Apyrases are characterized by their ability to hydrolyze both the ␥ -phosphate and  -phosphate on ATP and ADP by their need for divalent cations, and by their failure to be inhibited by any known class of phosphatase inhibitors (Plesner, 1995). Most apyrases are expressed as plasma membraneassociated proteins, with their hydrolytic activity facing out into the extracellular matrix (Wang and Guidotti, 1996). The apyrase we used is derived from pea and localizes in a manner consistent with other apyrases (Thomas et al., 1999). ...
... In Arabidopsis plants, the overexpression of a CD39 ectoapyrase homolog from pea (Wang and Guidotti, 1996) itself confers toxin resistance. This provides an example in which a multicellular organism has been genetically modified to have MDR addressed with a gene other than the one encoding an ABC transporter. ...
... The strongest known ATPases are apyrases, some of which have a turnover number of 10 4 molecules per sec (Handa and Guidotti, 1996). Because ectoapyrases are known to exist in human cells (Wang and Guidotti, 1996), we speculate that they could be the major ATPases on the cell surface. The ectoapyrase from pea plants, which is encoded by a single gene, accounts for Ͼ60% of the total cell surface ATPase activity in roots (Y. ...
Xenobiotic resistance in animals, plants, yeast, and bacteria is known to involve ATP binding cassette transporters that efflux invading toxins. We present data from yeast and a higher plant indicating that xenobiotic resistance also involves extracellular ATP degradation. Transgenic upregulation of ecto-ATPase alone confers resistance to organisms that have had no previous exposure to toxins. Similarly, cells that are deficient in extracellular ATPase activity are more sensitive to xenobiotics. On the basis of these and other supporting data, we hypothesize that the hydrolysis of extracellular ATP by phosphatases and ATPases may be necessary for the resistance conferred by P-glycoprotein.
... They usually have both ATPase and ADPase activities but their substrate speci®city varies broadly. CD39, the major ATPDase of vascular endothelial cells, has similar ADPase and ATPase activities (Kaczmarek et al., 1996; Wang & Guidotti, 1996; Marcus et al., 1997). The T-tubule enzyme is mainly an ATPase (Delgado et al., 1997). ...
... Under the conditions used, iodoacetamide had no action on apyrase activities. Endogenous ATPase activity of B10 cells was assessed using [g-32 P]-ATP as described previously (Wang & Guidotti, 1996). Brie¯y, cells grown in 60 mm Petri dishes were washed of their culture medium and further incubated at 378C into an Earle's salt solution supplemented with 0.1 mM ATP and 2 ± 4 mCi ml 71 [g-32 P]-ATP. ...
... Apyrase III has an ATPase to ADPase ratio of 6.2, similar to that of the T-tubule enzyme (Plesner, 1995). Apyrase VII has an ATPase to ADPase ratio of 0.7, similar to that of CD39 (Kaczmarek et al., 1996; Wang & Guidotti, 1996; Marcus et al., 1997).Figure 1 presents the results of a typical experiment in which cells were sequentially treated with ATP and apyrases. It shows that, as previously described (Hechler et al., 1998b), ATP, that had been cleaned of contaminating ADP, did not raise intracellular Ca 2+ . ...
ADP is a potent agonist of rat and human P2Y 1 purinoceptors. ATP is a weak competitive antagonist. This study analyses the situation in which P2Y 1 receptors are exposed to ATP in the presence of exogenous ecto‐nucleotidases (apyrases) that have high or low ATPase/ADPase activity ratio.
Rat brain capillary endothelial cells of the B10 clone express P2Y 1 receptors that couple to intracellular Ca ²⁺ mobilization. They have low endogenous ecto‐ATPase and ecto‐ADPase activities.
ATP did not raise intracellular Ca ²⁺ in B10 cells. Addition of apyrases III or VII (1 u ml ⁻¹ ) to ATP treated cells induced large intracellular Ca ²⁺ transients. Apyrases had no action in the absence of ATP.
A 1 u ml ⁻¹ apyrase III solution generated 20 μ M ADP from 0.1 m M ATP within 15 s. This concentration of ADP was sufficient to produce maximal activation of P2Y 1 receptors.
ATP was a full agonist of P2Y 1 receptors in the presence of 1 u ml ⁻¹ apyrase III. Dose response curves for the apparent actions of ATP were bell shaped in the presence of 0.1 u ml ⁻¹ apyrase III. Apyrase III did not alter ADP dose response curves when coincubated with ADP for 15 s.
Apyrase VII (1 u ml ⁻¹ ) shifted dose response curves for the actions of ADP to larger concentrations. It induced a bell shaped ATP dose response curve.
Results suggest that ATPDases prevent P2Y 1 receptor activation by degrading ADP but may contribute to P2Y 1 receptor activation by generating ADP from ATP.
British Journal of Pharmacology (1998) 125 , 675–680; doi: 10.1038/sj.bjp.0702115
... Apyrases are enzymes with the unifying characteristic of being able to hydrolyze both the ␥and the -phosphate on ATP or ADP (Plesner, 1995). Most apyrases are expressed as plasma membrane-associated proteins with their hydrolytic activity facing into the ECM (Wang and Guidotti, 1996). The pea (Pisum sativum) apyrase used in this investigation, psNTP9, was originally characterized as a 47-kD nuclear NTPase because it was initially purified from nuclei (Chen et al., 1987), because it was shown to be localized in the nucleus by immunocytochemistry (Tong et al., 1993), and because it had potential nuclear-localization and DNAbinding sequences (Hsieh et al., 1996). ...
... Many apyrases are ectoapyrases, which means that they are anchored in the plasma membrane with their active site facing into the ECM (Komoszynski and Wojtczak, 1996;Wang and Guidotti, 1996). Our results indicate that a significant fraction of the protein encoded by psNTP9 can also be classified as ectoapyrase. ...
ATP, which is present in the extracellular matrix of multicellular organisms and in the extracellular fluid of unicellular organisms, has been shown to function as a signaling molecule in animals. The concentration of extracellular ATP (xATP) is known to be functionally modulated in part by ectoapyrases, membrane-associated proteins that cleave the-and-phosphates on xATP. We present data showing a previously unreported (to our knowledge) linkage between apyrase and phosphate transport. An apyrase from pea (Pi-sum sativum) complements a yeast (Saccharomyces cerevisiae) phosphate-transport mutant and significantly increases the amount of phosphate taken up by transgenic plants overexpressing the gene. The transgenic plants show enhanced growth and augmented phosphate transport when the additional phosphate is supplied as inorganic phosphate or as ATP. When scavenging phosphate from xATP, apyrase mobilizes the-phosphate without promoting the transport of the purine or the ribose.
... Data are means ?SE of three determinations with different cell suspensions specificities and cellular locations (Zimmermann 1999). It has been demonstrated that mammalian membraneassociated ecto-ATPDase was homologous to human CD39, a lymphoid cell activation antigen (Wang and Guidotti 1996). Wang and Guidotti discovered that CD39 has sequence homology with a potato apyrase and that it exhibits apyrase activity (Wang and Guidotti 1996). ...
... It has been demonstrated that mammalian membraneassociated ecto-ATPDase was homologous to human CD39, a lymphoid cell activation antigen (Wang and Guidotti 1996). Wang and Guidotti discovered that CD39 has sequence homology with a potato apyrase and that it exhibits apyrase activity (Wang and Guidotti 1996). This work led to the identification of a family of ecto-ATPases that are related in sequence but vary in their membrane topology and tissue distribution (Plesner 1995;Zimmermann 1999;Goding 2000). ...
In this work we describe the ability of living Crithidia deanei to hydrolyze extracellular ATP. In intact cells at pH 7.2, a low level of ATP hydrolysis was observed in the absence of any divalent metal (0.41±0.13 nmol Pi h–1 107 cells–1). The ATP hydrolysis was stimulated by MgCl2 and the Mg2+-dependent ecto-ATPase activity was 4.05±0.17 nmol Pi h–1 107 cells–1. Mg2+-dependent ecto-ATPase activity increased linearly with cell density and with time for at least 60 min. The addition of MgCl2 to extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.93±0.26 mM MgCl2. This stimulatory activity was also observed when MgCl2 was replaced by MnCl2, but not CaCl2 or SrCl2. The apparent K
m for Mg-ATP2– was 0.26±0.03 mM. ATP was the best substrate for this enzyme; other nucleotides, such as ITP, GTP, UTP and CTP, produced lower reaction rates. In the pH range from 6.6 to 8.4, in which the cells were viable, the acid phosphatase activity also present in this cell decreased, while the Mg2+-dependent ATPase activity did not change. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A1, ouabain, vanadate, molybdate, sodium fluoride and tartrate. To confirm that this Mg2+-dependent ATPase was an ecto-ATPase, we used the impermeant inhibitor 4, 4′-diisothiocyanostylbene 2′-2′-disulfonic acid as well as suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg2+-dependent ATPase activity in a dose-dependent manner. The cell surface location of the ATP-hydrolyzing site was also confirmed by cytochemical analysis.
... We have already confirmed that the vascular ATPDase has common identity with the previously described B cell activation marker designated as CD39 (22); others have recently shown that CD39 has apyrase activity (47). This membrane protein, CD39, has several potential targets for oxidative damage since the protein is rich in cysteine, methionine, and tyrosine residues (22,47). ...
... We have already confirmed that the vascular ATPDase has common identity with the previously described B cell activation marker designated as CD39 (22); others have recently shown that CD39 has apyrase activity (47). This membrane protein, CD39, has several potential targets for oxidative damage since the protein is rich in cysteine, methionine, and tyrosine residues (22,47). ...
Quiescent endothelial cells (EC) regulate blood flow and prevent intravascular thrombosis. This latter effect is mediated in a number of ways, including expression by EC of thrombomodulin and heparan sulfate, both of which are lost from the EC surface as part of the activation response to proinflammatory cytokines. Loss of these anticoagulant molecules potentiates the procoagulant properties of the injured vasculature. An additional thromboregulatory factor, ATP diphosphohydrolase (ATPDase; designated as EC 3.6.1.5) is also expressed by quiescent EC, and has the capacity to degrade the extracellular inflammatory mediators ATP and ADP to AMP, thereby inhibiting platelet activation and modulating vascular thrombosis. We describe here that the antithrombotic effects of the ATPDase, like heparan sulfate and thrombomodulin, are lost after EC activation, both in vitro and in vivo. Because platelet activation and aggregation are important components of the hemostatic changes that accompany inflammatory diseases, we suggest that the loss of vascular ATPDase may be crucial for the progression of vascular injury.
... CD39 represents a marker of T-cell exhaustion that is predominantly observed in chronic viral infections, such as HIV and hepatitis B. The expression of CD39 on CD4+ T cells leads to a higher tendency to apoptosis; lower function of Fox+ regulatory T cells; and, by reducing the release of IL-21, also a lower activation of B cells, which limits T-cell functions in the acute phase of viral infections [29]. Furthermore, CD39 is also expressed on NK cells, and NKT cells regulate macrophage and monocyte activity and chemotaxis [28,30,[34][35][36]. ...
In patients with COVID-19, broad panels of immune checkpoint molecules (ICPMs) and the purinergic signaling have not been studied in parallel. We aimed to perform in-depth immunophenotyping of major cell subsets present in human peripheral blood of COVID-19 patients and controls using PD1, TIM3, LAG3, TIGIT, and CD200R, as well as CD39, as markers for the purinergic signaling pathway. We studied 76 COVID-19 patients and 12 healthy controls using peripheral blood mononuclear cells on flow cytometry. Univariable and multivariable statistics were performed. All ICPMs studied were significantly overexpressed on different cell subsets of COVID-19 patients when compared with healthy controls. Elevated lactate dehydrogenase; C-reactive protein; age; and high expression of CD45+, CD39+CD45+, TIM3+CD39+CD4+CD45+, and TIM3+CD39+CD8+CD3+CD4+ cells were significantly associated with severe COVID-19. On multivariable analysis, however, only high expression of CD39+CD45+ (OR 51.4, 95% CI 1.5 to 1763) and TIM3+CD39+CD4+CD3+CD45+ (OR 22.6, 95% CI 1.8 to 277) cells was an independent predictor for severe COVID-19. In conclusion, numerous ICPMs are overexpressed in COVID-19 patients when compared with healthy controls, suggesting a pathophysiological role of these molecules in SARS-CoV-2 infection. However, only TIM3 in co-expression with CD39 remained as a significant independent prognostic ICPM on multivariable analysis. The flow cytometric evaluation of TIM3+CD39+CD4+CD3+CD45+, as well as CD39+CD45+, is a powerful tool for the prognostication of COVID-19 patients on hospital admission.
... CD39 is an extracellular enzyme that converts extracellular ATP (eATP), a key activator of the NLRP3 inflammasome [21], to AMP [62]. A recent study used anti-CD39 Abs to confirm the relevance of inflammasome activation in tumor immunity triggered by PD-1 blockade [8]. ...
Recent studies have reported paradoxical roles of inflammation in tumor immunity triggered by PD-1 checkpoint antibody (Ab) blockade. Here, we elaborate on this controversy and propose a new perspective that might help understand this paradox. Since inflammatory cytokines and PD-1 blockade are known to target different subsets of exhausted CD8+ T cells, we propose that the timing at which anti-PD-1 Ab therapy and cytokine modulation occur might determine the fate of exhausted CD8+ T cells and perhaps, the clinical outcome of immunotherapeutic modalities.
... Alternatively, these enzymes can be immunohistochemically localised. Antibodies are available against CD39, an ecto-apyrase shown to be expressed in a variety of tissues, and linked to sites of purinergic signalling (Wang & Guidotti 1996& 1998, Vlajkovic et a l 1998. The presence of these enzymes on or around odontoblasts will provide further circumstantial evidence for the release of ATP from odontoblasts. ...
There is accumulating evidence that ATP may play a role in the initiation of pain by acting on P2X3 receptors expressed on nociceptive afferent nerve terminals. The aim of this study was to examine whether ATP and its receptors play a role in pain mechanisms in the human dental pulp by i) immunohistochemical staining of pulp tissue with antibodies to P2X3 receptors; and ii) by exploring whether odontoblasts release ATP. The presence, distribution and colocalisation of P2X3 receptors with other neural markers was investigated in human dental pulps. Pulps were removed from extracted third molars and stained with human anti-P2X3 antibody. P2X3 -immunoreactive (-ir) nerve fibres were detected in the main body of the pulp, in the subodontoblastic plexus of Raschkow and on nerve fibres projecting in between odontoblasts. Double labelling with anti-P2X3 and anti-neurofilament showed that the majority (but not all) fibres in the pulp, including both myelinated and unmyelinated nerve fibres, express P2X3 receptors. Double labelling with isolectin B4 (IB4) and anti-P2X3 showed that all P2X3-ir nerves also bind IB4. Therefore, both small and medium sized nerve fibres in the human pulp express P2X3 receptors. The possibility that odontoblasts could be potential sites for ATP release was studied using an in-situ method of odontoblast culture in 14 freshly extracted human third molars. Using this model, it was possible to directly stimulate the layer of odontoblasts still attached to dentin. The amount of ATP present in the solution bathing the odontoblasts was measured after stimulating the cells with hypo-osmotic solutions, ATP, and mechanical stimulation. Cell lysis was measured as each data point using a lactate dehydrogenase assay. Active ATP release was calculated by subtracting the ATP released as a result of cell lysis from the total bulk ATP. Between 0.5-10 nM active ATP release was observed when odontoblasts were stimulated by a 16-18% hyposmotic solution or by mechanical stimulation. These numbers, however, most probably under-represent the amount of actual ATP released during cell stimulation. In conclusion, P2X3 receptors are present on both myelinated and unmyelinated nerve fibres in human dental pulp, these receptors are probably responsive to ATP released from odontoblasts and may play a role in dental pain mechanisms. The roles of extracellular ATP in dental pulp are discussed.
... To mimic GPCR-mediated signaling events occurring in astrocytes in response to extracellular neurotransmitters and neuromodulators, Figueiredo et al. have expressed GPCRbased opsins, such as optoα1AR and optoβ2AR, in astrocytes to activate Gq-and Gs-mediated signaling cascades, respectively [107]. Photostimulation elicited an intracellular calcium increase in astrocytes expressing either opsin, which was blocked by apyrase, an enzyme hydrolyzing extracellular ATP, as well as by pharmacological blockers for the corresponding intracellular signaling cascade, such as inhibitors of phospholipase C and adenylate cyclase; these data indicate that a large portion of the calcium rise that was evoked by the activation of either opsin was a result of the autocrine action of extracellular ATP [108]. This study has demonstrated that GPCR-based opsins can be effectively used ...
The brain consists of heterogeneous populations of neuronal and non-neuronal cells. The revelation of their connections and interactions is fundamental to understanding normal brain functions as well as abnormal changes in pathological conditions. Optogenetics and chemogenetics have been developed to allow functional manipulations both in vitro and in vivo to examine causal relationships between cellular changes and functional outcomes. These techniques are based on genetically encoded effector molecules that respond exclusively to exogenous stimuli, such as a certain wavelength of light or a synthetic ligand. Activation of effector molecules provokes diverse intracellular changes, such as an influx or efflux of ions, depolarization or hyperpolarization of membranes, and activation of intracellular signaling cascades. Optogenetics and chemogenetics have been applied mainly to the study of neuronal circuits, but their use in studying non-neuronal cells has been gradually increasing. Here we introduce recent studies that have employed optogenetics and chemogenetics to reveal the function of astrocytes and gliotransmitters.
... Hydrolysis of ADP and UDP was also less than half of that for ATP. PeAPY2 219 activity was nearly undetectable for AMP (Fig. 3A), which was similar to 220 observations for other apyrases in plant and animal species (Wang and Guidotti, 1996; 221 Tanaka et al., 2011). 222 223 ...
Apyrase and extracellular ATP play crucial roles in mediating plant growth and defense responses. In the cold-tolerant poplar, Populus euphratica, low temperatures up-regulate apyrase (PeAPY2) expression in callus cells. We investigated the biochemical characteristics of PeAPY2 and its role in cold tolerance. We found that PeAPY2 predominantly localized to the plasma membrane, but punctuate signals also appeared in the endoplasmic reticulum and Golgi apparatus. PeAPY2 exhibited broad substrate specificity, but it most efficiently hydrolyzed purine nucleotides, particularly ATP. PeAPY2 preferred Mg2+ as a cofactor, and it was insensitive to various, specific ATPase inhibitors. When PeAPY2 was ectopically expressed in Arabidopsis, cold tolerance was enhanced, based on root growth measurements and survival rates. Moreover, under cold stress, PeAPY2-transgenic plants maintained plasma membrane integrity and showed reduced cold-elicited electrolyte leakage compared to wild type plants. These responses probably resulted from efficient plasma membrane repair via vesicular trafficking. Indeed, transgenic plants showed accelerated endocytosis and exocytosis during cold stress and recovery. We found that low doses of extracellular ATP accelerated vesicular trafficking, but high extracellular ATP inhibited trafficking and reduced cell viability. Cold stress caused significant increases in root medium extracellular ATP. However, under these conditions, PeAPY2-transgenic lines showed greater control of extracellular ATP levels than wild type plants. We concluded that Arabidopsis plants that overexpressed PeAPY2 could increase membrane repair by accelerating vesicular trafficking and hydrolyzing extracellular ATP to avoid excessive, cold-elicited ATP accumulation in the root medium, thus reduced ATP-induced inhibition of vesicular trafficking.
Copyright © 2015, Plant Physiology.
... Many cells express these enzymes at variable levels of activity, including neurones and leukocytes (Zimmerman, 1992; Ziganshin et al, 1994a ), with B lymphocytes showing higher ecto-ATPase and ecto-5′- nucleotidase activity than T lymphocytes (Barankiewicz et al., 1988). Recently, CD39, a marker expressed on activated lymphocytes, was identified as a major ecto-apyrase (ATP/ADPase) (Wang & Guidotti, 1996). Extracellular ATP may also be a substrate for ecto-protein kinases present on some cell types, including NK lymphocytes (Myers & Kang, 1990) and aortic endothelial cells (Pirotton et al, 1992). ...
... Moreover, it could possibly delay or even block the response process, via this giving an opportunity for these parasites to successfully establish the infection. As already known, the adenosine triphosphate (ATP) released by the injured host cells and tissues, as part of the pro-inflammatory process in which extracellular ATP (eATP), classified as a "Danger-Assosiated Molecular", acts via purinergic signaling to affect host immune response, this explained how the apyrase works during escaping the host immune system (Ting-Fang Wang 1996;Gounaris et al. 2004). All those mentioned above may reveal that in these parasites, the role of the apyrase is associated with virulence and the evasion of the parasite from the host defense mechanisms (D LeBel et al. 1980). ...
Apyrase encoding metal-ions activated plasma membrane protease is present in animal and plant tissues. This enzyme can hydrolyze ADP and ATP pyrophosphate bond, resulting in AMP and free phosphate groups, and plays an important role for insects and parasites to evade host immune system. However localization and function of apyrase in the canine hookworm, Ancylostoma caninum, remains unknown. To analyze apyrase gene in A. caninum (a eukaryotic parasitic hookworm), a pair of primers was designed according to the previous EST data. The full-length cDNA of apyrase gene was amplified from A. caninum by RT-PCR. The partial cDNA of apyrase encodes 249 amino acid protein was expressed in Escherechia coli. The recombinant protein was induced to express under proper conditions and the molecular size was as expected. The recombinant protein was purified. The transcripts of apyrase in different stages of A. caninum were analyzed by the Real-time PCR assay, and Immuno-localization assays were used to research the protein expression in different stages of A. caninum
... Analyses of expression of ecto-nucleotidases and adenosine receptors in C6Bu-1 cells by RT-PCR Recently, several ecto-enzymes involved in adenine nucleotide metabolism have been identi®ed and characterized. These include: the E-NTPase family, ecto-ATPase (CD39L1, Kegel et al. 1997) and ecto-apyrase (CD39, Wang and Guidotti 1996); the ecto-phosphodiesterase/ nucleotide pyrophosphatase (PDNP) family, PC-1 (PDNP1, van Driel et al. 1985), autotaxin (PDNP2, Murata et al. 1994) and GP130 RB13-6 (PDNP3, Deissler et al. 1995); and ecto-5 H -nucleotidase (also known as the lymphocyte membrane antigen CD73). To explore the ectonucleotidases involved in the hydrolysis of b,g-MeATP in C6Bu-1 cells, we designed the speci®c PCR primers for the six ecto-nucleotidases on the basis of rat cDNA nucleotide sequence of each enzyme. ...
The mechanism underlying β,γ-methylene ATP (β,γ-MeATP)-induced cAMP elevation was investigated in rat glioma C6Bu-1 cells. β,γ-MeATP increased forskolin-stimulated cAMP formation in a manner sensitive to both the P1 antagonist xanthine amine congener (XAC) and the P2 antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS). Adenosine deaminase (ADA; 1 U/mL), which abolished the adenosine-induced response, did not eliminate the β,γ-MeATP-induced response. However, combination of ADA with α,β-methylene ADP (α,β-MeADP), an ecto-5′-nucleotidase inhibitor, blocked the β,γ-MeATP-induced response. AMP, the substrate for ecto-5′-nucleotidase, also induced cAMP formation in a manner sensitive to XAC and α,β-MeADP inhibition. However, the AMP-induced response was not blocked by PPADS. HPLC analyses revealed that adenosine was generated from β,γ-MeATP and AMP. In addition, α,β-MeADP inhibited the conversion of β,γ-MeATP and AMP to adenosine, whereas PPADS blocked adenosine formation from β,γ-MeATP but not from AMP. [3H]Adenosine generated from [3H]AMP was preserved on the cell surface environment even in the presence of ADA. The mRNAs for ecto-phosphodiesterase/pyrophosphatase 1 (EC 3.1.4.1), ecto-5′-nucleotidase (EC 3.1.3.5) and adenosine A2B receptor were detected by RT-PCR. These results suggest that C6Bu-1 cells possess ecto-enzymes converting β,γ-MeATP to adenosine, and the locally accumulated adenosine in this mechanism efficiently stimulates A2B receptors in a manner resistant to exogenous ADA.
... We and others have shown that macrophages and microglial cells secrete ATP when stimulated with LPS (14,15,36), not to mention platelets that store massive amounts of ATP within dense granules (37), and can therefore cause significant increases in the extracellular ATP concentration when activated. That inflammatory cells are physiologically exposed to extracellular ATP is also hinted to by the expression of ATP-hydrolyzing enzymes, such as ecto-diphosphohydrolase (ecto-apyrase) and ecto-ATPase, on the outer aspect of their plasma membrane (38,39). ...
Endotoxin-dependent release of IL-1β from mouse microglial cells is a very inefficient process, as it is slow and leads to accumulation of a modest amount of extracellular cytokine. Furthermore, secreted IL-1β is mostly in the procytokine unprocessed form. Addition of extracellular ATP to LPS-primed microglia caused a burst of release of a large amount of processed IL-1β. ATP had no effect on the accumulation of intracellular pro-IL-1β in the absence of LPS. In LPS-treated cells, ATP slightly increased the synthesis of pro-IL-1β. Optimal ATP concentration for IL-1β secretion was between 3 and 5 mM, but significant release could be observed at concentrations as low as 1 mM. At all ATP concentrations IL-1β release could be inhibited by increasing the extracellular K+ concentration. ATP-dependent IL-1β release was also inhibited by 90 and 60% by the caspase inhibitors YVAD and DEVD, respectively. Accordingly, in ATP-stimulated microglia, the p20 proteolytic fragment derived from activation of the IL-1-β-converting enzyme could be detected by immunoblot analysis. These experiments show that in mouse microglial cells extracellular ATP triggers fast maturation and release of intracellularly accumulated IL-β by activating the IL-1-β-converting enzyme/caspase 1.
... The complement, density and distribution of enzymes are integral determinants of the breakdown rate and the relative concentrations of the mono-, di-, and triphosphate nucleotides in the extracellular space, reflecting in part different substrate affinities and end-products of the different enzymes (2,221,282). ENTPDase1 hydrolyzes ATP and ADP to AMP equally (117,264). In contrast, ENTPDase2 hydrolyzes ATP with far greater affinity than ADP and can cause the accumulation of ADP that favors the activation of P2YRs (136,175). ...
The main functions of the respiratory neural network are to produce a coordinated, efficient, rhythmic motor behavior and maintain homeostatic control over blood oxygen and CO2/pH levels. Purinergic (ATP) signaling features prominently in these homeostatic reflexes. The signaling actions of ATP are produced through its binding to a diversity of ionotropic P2X and metabotropic P2Y receptors. However, its net effect on neuronal and network excitability is determined by the interaction between the three limbs of a complex system comprising the signaling actions of ATP at P2Rs, the distribution of multiple ectonucleotidases that differentially metabolize ATP into ADP, AMP, and adenosine (ADO), and the signaling actions of ATP metabolites, especially ADP at P2YRs and ADO at P1Rs. Understanding the significance of purinergic signaling is further complicated by the fact that neurons, glia, and the vasculature differentially express P2 and P1Rs, and that both neurons and glia release ATP. This article reviews at cellular, synaptic, and network levels, current understanding and emerging concepts about the diverse roles played by this three-part signaling system in: mediating the chemosensitivity of respiratory networks to hypoxia and CO2/pH; modulating the activity of rhythm generating networks and inspiratory motoneurons, and; controlling blood flow through the cerebral vasculature. © 2013 American Physiological Society. Compr Physiol 3:331-363, 2013.
... CD39/NTPDase1 is a ubiquitously distributed acidic glycoprotein with a molecular mass of 70–100 kDa, which hydrolyzes ATP to ADP and subsequently to AMP868788 without substantial accumulation of ADP in the extracellular space [7] . CD39 was initially defined as a B-cell surface maturation marker [89]. ...
Extracellular nucleotides are ubiquitous signalling molecules which modulate distinct physiological and pathological processes. Nucleotide concentrations in the extracellular space are strictly regulated by cell surface enzymes, called ectonucleotidases, which hydrolyze nucleotides to the respective nucleosides. Recent studies suggest that ectonucleotidases play a significant role in inflammation by adjusting the balance between ATP, a widely distributed proinflammatory danger signal, and the anti-inflammatory mediator adenosine. There is increasing evidence for a central role of adenosine in alloantigen-mediated diseases such as solid organ graft rejection and acute graft-versus-host disease (GvHD). Solid organ and hematopoietic cell transplantation are established treatment modalities for a broad spectrum of benign and malignant diseases. Immunological complications based on the recognition of nonself-antigens between donor and recipient like transplant rejection and GvHD are still major challenges which limit the long-term success of transplantation. Studies in the past two decades indicate that purinergic signalling influences the severity of alloimmune responses. This paper focuses on the impact of ectonucleotidases, in particular, NTPDase1/CD39 and ecto-5'-nucleotidase/CD73, on allograft rejection, acute GvHD, and graft-versus-leukemia effect, and on possible clinical implications for the modulation of purinergic signalling after transplantation.
... At the maturation stage, GSS can stimulate 1-MeAde production in follicle cells regardless of the presence of AMP or adenosine. On the other hand, it was reported that mammalian and avian ATPDases show high homology with CD39, a molecule originally characterized as a lymphoid surface antigen (Handa and Guidotti, 1996; Kaczmarek et al., 1996; Wang and Guidotti, 1996). Since each oocyte in starfish ovaries is surrounded by follicle cells (Schroeder, 1971; Schroeder et al., 1979 ), ecto- ATPDase on starfish ovarian follicle cells may protect oocytes from phagocytes and parasites during oogenesis. ...
A high activity of ecto-ATP diphosphohydrolase (ATPDase, EC 3.6.1.5) is present on the surface of ovarian follicle cells of the starfish Asterina pectinifera. It is known that follicle cells play an important role in oocyte maturation by producing 1-methyladenine (1-MeAde) as the maturation-inducing substance. This study was undertaken to determine if ecto-ATPDase is associated with 1-MeAde production. 1-MeAde production is induced by a gonad-stimulating substance (GSS) secreted from nervous tissue. It has also been shown that G-proteins and adenylyl cyclase are involved in the action of GSS in 1-MeAde production. When isolated follicle cells were incubated with the ecto-ATPDase metabolites, adenosine and AMP, a slight decrease in intracellular cyclic AMP level was observed. Adenosine also inhibited adenylyl cyclase in the presence of GTP. Pertussis toxin reversed adenosine-induced inhibition, suggesting that adenosine acts via the Gi receptor. On the other hand, GSS could induce 1-MeAde production and an increase in cyclic AMP level regardless of the absence or presence of AMP or adenosine. GSS-dependent 1-MeAde production markedly decreased after washing of follicle cells with seawater containing low concentrations of Ca2+, but the activity of ecto-ATPDase remained constant. Neither NaF nor adenylimidodiphosphate, which are inhibitors of ecto-ATPDase, had any effect on GSS-induced 1-MeAde production. Thus, it is unlikely that ecto-ATPDase is directly involved in 1-MeAde production in starfish ovarian follicle cells. Since an increase in cyclic AMP level is indispensable for 1-MeAde production, it may be possible that adenosine and AMP produced by ecto-ATPDase play a role in prevention of precocity before oocyte maturation stage.
... There are now a large number of ecto-enzymes that are capable of degrading ATP (94,95). There is considerable evidence for a role for the ecto-phosphodiesterases in the recycling and uptake of extracellular nucleotides (67). ...
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 enzymes differ regarding their preference for the hydrolysis of nucleoside 5¢-tri-and diphosphates. NTPDase1 (CD39) hydrolyses nucleoside 5¢-triphosphates and -diphosphates equally well (Kaczmarek et al., 1996; Wang & Guidotti, 1996); NTPDase3 (HB6) has a threefold preference for nucleoside 5¢- triphosphates over -diphosphates (Smith & Kirley, 1998), whilst NTPDase2 (ecto-ATPase) hydrolyses nucleoside 5¢-diphosphates only to a marginal extent (Kegel et al., 1997; Heine et al., 1999). The expression pattern of the individual ecto-nucleotidases and their function-related cellular distribution has not been clari®ed to date. ...
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.
... Given that Luo et al. observed a selective expression of the uridine nucleotide-sensitive receptors (P2Y 2 and P2Y 4 ) in the apical membranes of the ductal epithelial cells, it is interesting to speculate whether UTP might be preferentially released into the luminal compartment under some conditions. It should be noted that extracellular nucleotide tri-and diphosphates can be rapidly metabolized by CD39 family ectoapyrases to nucleotide monophosphates (20). In turn, extracellular monophosphates can be hydrolyzed to their respective nucleosides (AMP to adenosine and UMP to uridine) by the CD73 5Ј-ectonucleotidase that is highly expressed in the apical membranes of many epithelial cells (21). ...
... We and others have shown that macrophages and microglial cells secrete ATP when stimulated with LPS (14,15,36), not to mention platelets that store massive amounts of ATP within dense granules (37), and can therefore cause significant increases in the extracellular ATP concentration when activated. That inflammatory cells are physiologically exposed to extracellular ATP is also hinted to by the expression of ATP-hydrolyzing enzymes, such as ecto-diphosphohydrolase (ecto-apyrase) and ecto-ATPase, on the outer aspect of their plasma membrane (38,39). ...
... Nucleoside triphosphate diphosphohydrolases, also called NTDPases or apyrases, are ubiquitous enzymes that hydrolyze nucleoside 5 0 -triphosphates and 5 0 -diphosphates with varying nucleotide preferences and in the presence of divalent cations. They are usually integral membrane proteins, and have roles in a variety of biological processes (Wang & Guidotti, 1996;Kegel et al., 1997;Zimmermann, 2000). ...
The Kluyveromyces lactis ORF r_klactIV3,463 on chromosome IV, hereafter named KlYND1, encodes an endoapyrase that has nucleoside phosphatase activity with a lumenal orientation. The enzyme showed equally high activity towards GDP/UDP and ADP, and also showed activity, although to a lesser extent, towards GTP. No activity was detected with the other triphosphates and all monophosphates. The overexpression of KlYND1 in Klgda1Delta cells of K. lactis, devoid of the encoded GDPase/UDPase activity, suppressed the loss of O-glycosylation and cell wall-related defects described in such mutants, and suggests a partial overlap of function between the two genes, and therefore some redundancy. The overexpression of KlYND1 in wild-type cells enhanced the secretion of the recombinant human serum albumin and glucoamylase employed as reporters.
... C-1 is coordinately up-regulated in activated T cells with two other ectoenzymes involved in nucleotide metabolism, CD38 NAD glycohydrolase and CD73 5-nucleotidase (21). Autotaxin is able to hydrolyze ATP to ADP or AMP and AMP to adenosine (12). It is, therefore, able to perform the functions of CD39 apyrase, an ATP diphosphohydrolase (E.C. 3.6.1.5 [68] ), and CD73 ecto- 5-nucleotidase (E.C. 3.1.3.5 [62] ), converting ATP to adenosine , which can be taken up by cells (11). Acting alone or in concert, these enzymes may provide cells with external supplementary sources of nucleosides. The presence in the FWPV genome of thymidine and thymidylate kinases indicates that the intracellular si ...
The genome of fowlpox virus (FWPV), type species of the Avipoxviridae, is considerably rearranged compared with that of vaccinia virus (the prototypic poxvirus and type species of the Orthopoxviridae) and is 30% larger. It is likely that the genome of FWPV contains genes in addition to those found in vaccinia virus, probably involved with its replication and survival in the chicken. A 7,470-bp segment of the FWPV genome has five open reading frames (ORFs), two of which encode ankyrin repeat proteins, many examples of which have been found in poxviruses. The remaining ORFs encode homologs of cellular genes not reported in any other virus. ORF-2 encodes a homolog of the yeast Sec17p and mammalian SNAP proteins, crucial to vesicular transport in the exocytic pathway. ORF-3 encodes a homolog of an orphan human protein, R31240_2, encoded on 19p13.2. ORF-3 is also homologous to three proteins (YLS2, YMV6, and C07B5.5) from the free-living nematode Caenorhabditis elegans and to a 43-kDa antigen from the parasitic nematode Trichinella spiralis. ORF-5 encodes a homolog of the mammalian plasma cell antigen PC-1, a type II glycoprotein with exophosphodiesterase activity. The ORFs are present in the virulent precursor, HP1, of the sequenced attenuated virus (FP9) and are conserved in other strains of FWPV. They were shown, by deletion mutagenesis, to be nonessential to virus replication in tissue culture. RNA encoding the viral homolog of PC-1 is expressed strongly early and late in infection, but RNAs encoding the homologs of SNAP and R31240_2 are expressed weakly and late.
... When ADP was applied as a substrate for the same period of time as ATP, the general histochemical staining was less intense than that for ATP (Fig. 4a–e). As expected for the distribution of NTPDase1, which hydrolyzes ADP and ATP almost equally well (0.6:1 to 0.9:1; Wang and Guidotti 1996; Heine et al. 1999), microglia and blood vessels were clearly depicted by ADPase histochemistry and accounted for much of the staining in the sections (see Comparisons below). Since NTPDase2 hydrolyzes ADP only to a minor extent (Heine et al. 1999), elevated staining was absent from the neurogenic regions: the subventricular zone, the RMS of the olfactory bulb, and the subgranular layer of the dentate gyrus (Fig. 4b; see also Comparisons below). ...
Nucleotides comprise a major class of signaling molecules in the nervous system. They can be released from nerve cells, glial cells, and vascular cells where they exert their function via ionotropic (P2X) or metabotropic (P2Y) receptors. Signaling via extracellular nucleotides and also adenosine is controlled and modulated by cell-surface-located enzymes (ectonucleotidases) that hydrolyze the nucleotide to the respective nucleoside. Extracellular hydrolysis of nucleotide ligands involves a considerable number of enzymes with differing catalytic properties differentially affecting the nucleotide signaling pathway. It is therefore important to investigate which type of ectonucleotidase(s) contributes to the control of nucleotide signaling in distinct cellular and physiological settings. By using a classical enzyme histochemical approach and employing various substrates, inhibitors, and knockout animals, we provide, for the first time, a comparative analysis of the overall distribution of catalytic activities reflecting four ectonucleotidase families: ecto-5'-nucleotidase, alkaline phosphatases, ectonucleoside triphosphate diphosphohydrolases (E-NTPDases), and ectonucleotide pyrophyphatases/phosphodiesterases (E-NPPs). We place into perspective the earlier literature and provide novel evidence for a parenchymal localization of tissue non-specific alkaline phosphatase, E-NPPs, and E-NTPDases in the mouse brain. In addition, we specify the location of ectonucleotidases within the brain vasculature. Most notably, brain vessels do not express ecto-5'-nucleotidase. The preponderance of individual enzymes differs considerably between brain locations. The contribution of all types of ectonucleotidases thus needs to be considered in physiological and pharmacological studies of purinergic signaling in the brain.
The aim of this thesis was to isolate transcribed sequences from the Nevoid Basal Cell Carcinoma Syndrome (NBCCS) candidate region on human chromosome 9q22. We have used the technique of cDNA selection to isolate transcribed sequences from a minimal set of cosmid and PAC clones, covering approximately 1.5 megabases of DNA from 9q22. Twenty seven putative transcripts were identified, and five further characterised. A new member of the forkhead gene family was isolated, FKHL15. A repeat of 19 alanine residues was found in FKHL15, encoded in part by a trinucleotide repeat. Polyalanine stretches have been shown to be associated with transcriptional repression. A human and mouse gene that showed high amino acid identity to the rat ninjurin protein, were cloned and mapped. Rat ninjurin has been shown to be involved in homotypic fusion and the repair of damaged sensory neurones. A novel PHD finger containing gene was isolated, GRC-5. The PHD finger is a member of the Zn-finger family of motifs, and may be involved in protein- protein or protein-DNA interactions. A human gene showing high amino acid homology to the lymphoid cell activation antigen CD39 was cloned and mapped to 9q34(CD39L1). Database searches with the CD39L1 protein sequence revealed that it is likely to be a member of the ectoATPase gene family, and that several other human homologues exist. A small gene (GRC-7), was isolated and appears to encode a novel protein with no homology to entries in any database. The underlying genetic defect in patients with NBCCS was recently shown to be due to mutations in the human homologue of the drosophila segment polarity gene patched. Two other human diseases show linkage to genetic markers on human chromosome 9q22; Hereditary Sensory Neuropathy Type I (HSN-I) and Multiple Self Healing Squamous Epitheliomata (ESS1). The etiology of HSN-I and ESS1 are unknown, and the above genes are candidates.
Dans le cerveau, les fonctions de la phosphatase alcaline non spécifique des tissus (TNAP) ne sont pas clairement identifiées. La localisation et l'expression de cette enzyme au niveau neuronal suggère cependant, qu'elle joue un rôle important dans le développement et le fonctionnement du cerveau. Cela est supporté par la présence de graves crises d'épilepsie chez les humains porteurs d'une mutation de la TNAP. Ces crises d'épilepsie sont létales chez les souris KO pour la TNAP. Des études chez la souris montrent que la TNAP pourrait réguler l'inhibition postsynaptique médiée par le GABA et elle pourrait être impliquée dans l'inhibition présynaptique médiée par l'adénosine. L'adénosine est, en partie, synthétisée via la déphosphorylation successive de l'ATP en ADP puis en AMP par des ectonucléotidases. Parmi elles, la TNAP et l'ecto- 5'-nucléotidase (NT5E) catalysent l'hydrolyse de l'AMP en adénosine dans le cortex cérébral. L'adénosine agit principalement au niveau présynaptique par l'intermédiaire des récepteurs A1. Ainsi l'adénosine a une influence sur la transmission synaptique et sur la plasticité synaptique. Ceci pourrait expliquer, en partie, les crises d'épilepsie observées chez les souris KO pour la TNAP. Les deux objectifs principaux de ma thèse ont été : (1) évaluer la contribution de la TNAP dans la production d'adénosine dans le cerveau ; (2) étudier l'influence de l'adénosine sur la plasticité synaptique. Premièrement, l'étude de la contribution de la TNAP dans la production d'adénosine dans le cerveau a été réalisée au moyen de deux approches complémentaires. Une approche métabolomique (spectroscopie RMN du proton) sur des cerveaux entiers de souris KO pour la TNAP a permis de montrer que la TNAP participe, entre autre, à la synthèse d'adénosine dans le cerveau. Une deuxième approche, électrophysiologique sur tranches de cerveaux de souris in vitro, nous permet d'examiner les conséquences de l'inhibition des ectonucléotidases intervenant dans la synthèse de l'adénosine. Elle a révélé que l'inhibition des ectonucléotidases (TNAP et NT5E) ne supprime pas l'effet inhibiteur de l'AMP médiée par les récepteurs A1. Deuxièmement, nous avons étudié l'influence de l'adénosine sur la plasticité synaptique à courte terme. Nous avons enregistré des potentiels de champs dans la couche Ia du cortex piriforme en réponse à des stimulations électriques (3,125 à 100 Hz) présentée avec des fréquences recouvrant la gamme d'oscillations physiologiques. Nos résultats montrent qu'avec de fortes concentrations d'adénosine, la facilitation est accentuée par rapport à celle observée en situation contrôle. Cet effet est observé pour des fréquences supérieures ou égales à 25 Hz. De plus, cette accentuation est d'autant plus grande que la fréquence est élevée (maximum atteint à 100 Hz pour 100 µM). En bloquant l'action de l'adénosine endogène, l'effet contraire est observé : une facilitation déficitaire par rapport au contrôle et dont le défaut est croissant avec la fréquence de stimulation. Tous ces résultats convergent vers l'hypothèse qu'une déficience en TNAP, traduite par une absence d'adénosine, pourrait contribuer au maintien des processus épileptiques générés par un déséquilibre de l'inhibition et de l'excitation dû à une diminution de GABA. L'effet inhibiteur de l'AMP médié par les récepteurs A1 ne serait pas suffisant pour contrecarrer les crises d'épilepsie observées chez les sujets hypophosphatasiques et les souris KO pour la TNAP.
Background:
Microglia play vital roles in neurotrophic support and modulating immune or inflammatory responses to pathogens or damage/stressors during disease. This study describes the ability to establish large numbers of microglia from embryonic tissues with the addition of granulocyte-macrophage stimulating factor (GM-CSF) and characterizes their similarities to adult microglia examined ex vivo as well as their responses to inflammatory mediators.
Method:
Microglia were seeded from a primary embryonic mixed cortical suspension with the addition of GM-CSF. Microglial expression of CD45, CD11b, CD11c, MHC class I and II, CD40, CD80, and CD86 was analyzed by flow cytometry and compared to those isolated using different culture methods and to the BV-2 cell line. GM-CSF microglia immunoreactivity and cytokine production was examined in response to lipopolysaccharide (LPS) and interferon-γ (IFN-γ).
Results:
Our results demonstrate GM-CSF addition during microglial culture yields higher cell numbers with greater purity than conventionally cultured primary microglia. We found that the expression of immune markers by GM-CSF microglia more closely resemble adult microglia than other methods or an immortalized BV-2 cell line. Primary differences amongst the different groups were reflected in their levels of CD39, CD86 and MHC class I expression. GM-CSF microglia produce CCL2, tumor necrosis factor-α, IL-6 and IL-10 following exposure to LPS and alter costimulatory marker expression in response to LPS or IFN-γ. Notably, GM-CSF microglia were often more responsive than the commonly used BV-2 cell line which produced negligible IL-10.
Conclusion:
GM-CSF cultured microglia closely model the phenotype of adult microglia examined ex vivo. GM-CSF microglia are robust in their responses to inflammatory stimuli, altering immune markers including Iba-1 and expressing an array of cytokines characteristic of both pro-inflammatory and reparative processes. Consequently, the addition of GM-CSF for the culturing of primary microglia serves as a valuable method to increase the potential for studying microglial function ex vivo.
Background: Diabetes mellitus (DM) patients lose their ability to control normal blood glucose levels, resulting in high blood glucose levels (hyperglycemia). Hyperglycemia causes DM complications. This involves responses of vascular endothelial cells (VECs) to hyperglycemia, affecting inflammatory process and platelet activity. Ecto-enzyme CD39 is expressed on VECs, catalyzing the hydrolysis of ATP and ADP to AMP and, consequently, regulating inflammatory process and platelet activation.
Objective: We studied whether high glucose concentration has an effect on CD39 expression on VECs.
Methods: Cultured human umbilical vein endothelial cells (HUVEC) were used as a model of study. HUVEC were cultured in different glucose conditions (4, 9, 24, and 34 mM) for 24 hours. Cell viability was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based assay and expression of CD39 was examined by using SDS-PAGE and western blot techniques.
Results: HUVEC were cultured in normal (4 and 9 mM) or high (24 and 34 mM) glucose concentrations for short term (24 hours). The results showed that high glucose (24 and 34 mM) reduced cell viability to 89.5 ± 11.3 and 86.3 ± 13.5 (mean ± SD), compared with control (4 mM), respectively. High glucose also induced increases in CD39 expression in HUVEC.
Conclusion: High glucose decreases cell viability and increases CD39 expression in HUVEC, suggesting involvement of CD39 in cell responses to high glucose.
The modulatory role of extracellular nucleotides and adenosine in relevance to purinergic cell signaling mechanisms has long been known and is an object of much research worldwide. These extracellular nucleotides are released by a variety of cell types either innately or as a response to patho-physiological stress or injury. A variety of surface-located ecto-nucleotidases (of four major types; nucleoside triphosphate diphosphohydrolases or NTPDases, nucleotide pyrophosphatase/phosphodiesterases or NPPs, alkaline phosphatases APs or ALPs, and ecto-5'-nucleotidase or e5NT) are responsible for meticulously controlling the availability of these important signaling molecules (at their respective receptors) in extracellular environment and are therefore crucial for maintaining the integrity of normal cell functioning. Overexpression of many of these ubiquitous ecto-enzymes has been implicated in a variety of disorders including cell adhesion, activation, proliferation, apoptosis, and degenerative neurological and immunological responses. Selective inhibition of these ecto-enzymes is an area that is currently being explored with great interest and hopes remain high that development of selective ecto-nucleotidase inhibitors will prove to have many beneficial therapeutic implications. The aim of this review is to emphasize and focus on recent developments made in the field of inhibitors of ecto-nucleotidases and to highlight their structure activity relationships wherever possible. Most recent and significant advances in field of NTPDase, NPP, AP, and e5NT inhibitors is being discussed in detail in anticipation of providing prolific leads and relevant background for research groups interested in synthesis of selective ecto-nucleotidase inhibitors.
Extracellular nucleotides are involved in the control of mucociliary clearance (MCC) in human airways. Because of their therapeutic potential for obstructive lung diseases such as cystic fibrosis (CF), we investigated nucleotide metabolism on airway epithelial surfaces. The mucosal surface of human nasal and bronchial epithelial cells in culture dephosphorylated ATP into ADP, AMP, and adenosine. Bronchial cells hydrolyzed ATP at a faster rate than nasal cells, 3.8 ± 0.5 nmol·min–1 cm–2 and 2.2 ± 0.4 nmol·min–1 cm–2, respectively. The ratios of ATP/ADP hydrolysis for bronchial and nasal epithelia were 2.9 ± 0.6 and 2.1 ± 0.4, respectively. Divalent cations were required for ATP hydrolysis (Ca2+ > Mg2+) and pH dependency profile revealed two peaks, at pH 7.5 ad 9.0. Kinetic analysis supported the coexpression of more than one ATP-hydrolyzing activity on nasal (Km = 17 ± 2 μM, 129 ± 5 μM, and 405 ± 12 μM) and bronchial (Km = 12 ± 5 μM and 136 ± 8 μM) epithelial cells. Reverse-transcriptase polymerase chain reaction confirmed the coexpression of all three families of ATP-hydrolyzing ectoenzymes: ecto-nucleoside triphosphate diphosphohydrolases, ecto-nucleotide pyrophosphatase/phosphodiesterases, and alkaline phosphatases. Altogether, these findings demonstrate the presence of a multienzyme system in human airways, with the capacity to control ATP concentrations under physiological and pathological conditions. The identification of the major enzyme(s) responsible for ATP hydrolysis in human airways may lead to the development of specific inhibitors that would improve nucleotide-based treatments of MCC in patients with CF. Drug Dev. Res. 52:66–75, 2001. © 2001 Wiley-Liss, Inc.
The effect of extracellular ATP on the activation of NFAT, an important transcription factor involved in cytokine expression, was investigated in the mouse microglial cell line N9. Incubation with ATP resulted in the potent and rapid activation of NFAT. Both NFAT1 and NFAT2 were activated by a calcineurin-dependent pathway that required the influx of extracellular Ca2+. Inhibition of calcineurin by immunosuppressive drugs or Ca2+ chelation prevented ATP-induced activation and nuclear translocation of NFAT. Activation of NFAT by ATP involved a P2X7 purinoreceptor-mediated pathway. 2′,3′ -(4-benzoyl)-benzoyl-ATP, an ATP analog with high affinity to the P2X7 receptor, was a strong agonist of NFAT activation. In addition, microglial N9 clones selected for the absence of P2X7, but still expressing the P2Y subtype, revealed no NFAT activation in response to ATP, although NFAT could be still triggered by agents increasing intracellular Ca2+. Our data therefore suggest that triggering of the P2X7 receptor by extracellular ATP is a potent mediator of transcription factor NFAT activation, and thus could be involved in early proinflammatory processes in the immune and nervous system. Drug Dev. Res. 52:213–219, 2001. © 2001 Wiley-Liss, Inc.
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.
Microglial cells represent resident macrophages of the central nervous system where they resume a resting state of unknown physiological function. They are rapidly transformed into an activated state by acute pathological events affecting the nervous system. Activated microglia function in tissue repair and regeneration but may also enhance tissue damage. Cultured microglial cells express both P2Y and P2X receptors. Activation of the microglial P2X7 receptor can result in the release of cytokines or also mediate cytotoxicity in microglial cells. Microglial cells have developed mechanisms for controlling the surrounding concentrations of free extracellular ATP via a surface-expressed ectonucleotidase chain. We analyzed the expression of the ectonucleotidases associated with resting microglia and microglia activated by two models of cerebral ischemia. Out of a plethora of enzymes with the potential to hydrolyze extracellular nucleotides, the major ectonucleotidases associated with microglia are NTPDase1, which hydrolyzes ATP and ADP, and ecto-5′-nucleotidase, which catalyzes the hydrolysis of AMP to adenosine. This is shown by enzyme histochemical analysis, immunocytochemistry, Northern hybridization, and the analysis of NTPDase1-deficient mice. Our results suggest that a major function of resting microglia could be in the lowering of interstitial levels of ATP acting either via autocrine pathways or in the ATP-involving signaling pathways between adjacent neurons and astrocytes. Enhanced activity of NTPDase1 may protect microglia, activated by various pathological settings, from overstimulation by ATP released from the injured tissue. The parallel increase in activity of ecto-5′-nucleotidase would facilitate the formation of the final hydrolysis product adenosine that exerts neuromodulatory and immunomodulatory actions and contributes to the protection of neurons. Drug Dev. Res. 53:208–217, 2001. © 2001 Wiley-Liss, Inc.
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.
In this study, we have investigated the distribution of the enzyme nucleoside triphosphate diphosphohydrolase-1 (NTPDase1; EC 3.6.1.5) in a subset of pig tissues by biochemical activity and Western blotting with antibodies against porcine NTPDase1. The highest expression of this enzyme was found in vascular endothelium, smooth muscle, spleen and lung.
The complete cDNA of NTPDase1 from aorta endothelial cells was sequenced using primer walking. The protein consists of 510 amino acids, with a calculated molecular mass of 57 756 Da. The amino-acid sequence indicated seven putative N-glycosylation sites and one potential intracellular cGMP- and cAMP-dependent protein kinase phosphorylation site. As expected, the protein has a very high homology to other known mammalian ATPDases and CD39 molecules, and includes all five apyrase conserved regions.
Expression of the complete cDNA in COS-7 cells confirmed that NTPDase1 codes for a transmembrane glycoprotein with ecto-ATPase and ecto-ADPase activities. Two proteolytic products of NTPDase1, with molecular mass of 54 and 27 kDa, respectively, were consistently present in proteins from transfected COS-7 cells and in particulate fractions from different tissues. A trypsin cleavage site, giving rise to these two cleavage products, was identified. In order to remain enzymatically active, the two cleavage products have to interact by non–covalent interactions.
Nucleoside triphosphate diphosphohydrolases 1, 2, 3 and 8 (NTPDases 1, 2, 3 and 8) are the dominant ectonucleotidases and thereby expected to play important roles in nucleotide signaling. Distinct biochemical characteristics of individual NTPDases should allow them to regulate P2 receptor activation differentially. Therefore, the biochemical and kinetic properties of these enzymes were compared. NTPDases 1, 2, 3 and 8 efficiently hydrolyzed ATP and UTP with K
m values in the micromolar range, indicating that they should terminate the effects exerted by these nucleotide agonists at P2X1- and P2Y2,4,11 receptors. Since NTPDase1 does not allow accumulation of ADP, it should terminate the activation of P2Y1,12,13 receptors far more efficiently than the other NTPDases. In contrast, NTPDases 2, 3 and 8 are expected to promote the activation of ADP specific receptors, because in the presence of ATP they produce a sustained (NTPDase2) or transient (NTPDases 3 and 8) accumulation of ADP. Interestingly, all plasma membrane NTPDases dephosphorylate UTP with a significant accumulation of UDP, favoring P2Y6 receptor activation. NTPDases differ in divalent cation and pH dependence, although all are active in the pH range of 7.0-.5. Various NTPDases may also distinctly affect formation of extracellular adenosine and therefore adenosine receptor-mediated responses, since they generate different amounts of the substrate (AMP) and inhibitor (ADP) of ecto-5-nucleotidase, the rate limiting enzyme in the production of adenosine. Taken together, these data indicate that plasma membrane NTPDases hydrolyze nucleotides in a distinctive manner and may therefore differentially regulate P2 and adenosine receptor signaling.
Ecto-nucleotidases play a pivotal role in purinergic signal transmission. They hydrolyze extracellular nucleotides and thus can control their availability at purinergic P2 receptors. They generate extracellular nucleosides for cellular reuptake and salvage via nucleoside transporters of the plasma membrane. The extracellular adenosine formed acts as an agonist of purinergic P1 receptors. They also can produce and hydrolyze extracellular inorganic pyrophosphate that is of major relevance in the control of bone mineralization. This review discusses and compares four major groups of ecto-nucleotidases: the ecto-nucleoside triphosphate diphosphohydrolases, ecto-5'-nucleotidase, ecto-nucleotide pyrophosphatase/phosphodiesterases, and alkaline phosphatases. Only recently and based on crystal structures, detailed information regarding the spatial structures and catalytic mechanisms has become available for members of these four ecto-nucleotidase families. This permits detailed predictions of their catalytic mechanisms and a comparison between the individual enzyme groups. The review focuses on the principal biochemical, cell biological, catalytic, and structural properties of the enzymes and provides brief reference to tissue distribution, and physiological and pathophysiological functions.
Recent findings indicate that extracellular ATP (xATP) plays an important regulatory role in both animals and plants. The turnover of xATP is controlled largely by the activity of ecto-phosphatases, including ecto-apyrases. Two apyrases, termed Atapy1 (Arabidopsis thaliana apyrase 1) and Atapy2, were cloned and sequenced. The transcripts of Atapy1 and Atapy2 are widely distributed; however, the expression patterns are not identical. In roots, for example, the mRNA level of Atapy1 is greater than that of Atapy2. Atapy1 and Atapy2 are 87 % identical at the amino acid sequence level. Both contain four apyrase conserved regions (ACRs), an ATP-binding motif and a hydrophobic segment at the N-terminus. However, only Atapy1 demonstrates a calmodulin (CaM)-binding domain. The two cDNAs were overexpressed in bacteria, and the resulting proteins were biochemically analyzed. The purified proteins displayed enzymatic properties characteristic of apyrases, such as the hydrolysis of ATP and ADP, but not AMP, and an insensitivity to inhibitors of ATPases. In a CaM-binding assay, only the protein of Atapy1 bound to CaM under the conditions tested. To date Atapy1 represents the only other apyrase, besides pea NTPase, shown to contain a functional CaM-binding domain.
Human soluble calcium-activated nucleotidase (human SCAN) is a homologue of the salivary anti-coagulant apyrases injected by insects into their hosts to allow blood feeding. However, the human enzyme, unlike its insect counterparts, does not efficiently hydrolyze the platelet agonist, ADP. By site-directed mutagenesis, two mutant human SCANs were constructed and expressed in bacteria. Following refolding from inclusion bodies and purification, these enzymes were assessed for anti-coagulant and anti-thrombotic efficacy. These engineered proteins include both active site mutations and a dimer interface mutation to increase the stability and ADPase activity of the modified human nucleotidase. The ADPase activity of these mutants increased more than ten fold. The E130Y/K201M/E216M SCAN mutant efficiently inhibited platelet aggregation in vitro. In addition, the E130Y/K201M/T206K/T207E/E216M mutant inhibited jugular vein thrombosis in the murine ferric chloride-induced model of thrombosis, as assessed by laser Doppler blood flow measurements. The bed bug insect homologue of human SCAN was also expressed and purified, and used in these in vivo experiments as a benchmark to assess the therapeutic potential of the engineered human enzymes. The most active modified human enzyme was able to completely inhibit the thrombosis induced by ferric chloride at roughly double the protein dose used for the bed bug enzyme. Thus, for the first time, we show that an engineered form of this human protein is efficacious in an in vivo model of thrombosis, demonstrating that suitably modified human SCAN enzymes have therapeutic potential as anti-coagulant and anti-thrombotic therapeutic agents. This suggests their utility in future treatment strategies for thrombotic cardiovascular diseases, including myocardial infarctions and ischemic strokes.
The role of extracellular purines and purinoreceptors in the pathophysiology of different neurological disorders is the focus of rapidly expanding area of research. Ectonucleotidases are the enzymes with multiple roles in extracellular nucleotides metabolism and regulation of nucleotidebased intercellular signaling. The aim of present study was to investigate the changes in the ATP, ADP and AMP hydrolyzing activities after ribavirin treatment in spinal cord during experimental autoimmune encephalomyelitis (EAE). Our results demonstrate that ribavirin itself had no significant effect on ectoenzyme activities, when tested in vitro and in vivo on spinal cord crude membrane preparation of intact animals. We observed significant increase in ATP, ADP and AMP hydrolyzing activity in the spinal cord crude membrane preparation in EAE animals at 15 days post immunization compared to control animals. The increase was registered at 28 days post immunization, as well. At same time points, ribavirin treatment decreased ATP, ADP and AMP hydrolyzing activity compared to EAE animals. In addition, no significant changes 8 days post immunization was observed between EAE-induced and ribavirin- treated EAE animals and these levels were similar to control level. Thus, we suppose that ribavirin-induced alteration in ectonucleotidase activities is rather due to its suppression of inflammation, than to its direct action on ATP, ADP and AMP hydrolysis.
The first comprehensive review of the ubiquitous "ecto-ATPases" by Plesner was published in 1995. A year later, a lymphoid cell activation antigen, CD39, that had been cloned previously, was shown to be an ecto-ATPase. A family of proteins, related to CD39 and a yeast GDPase, all containing the canonical apyrase conserved regions in their polypeptides, soon started to expand. They are now recognized as members of the GDA1_CD39 protein family. Because proteins in this family hydrolyze nucleoside triphosphates and diphosphates, a unifying nomenclature, nucleoside triphosphate diphopshohydrolases (NTPDases), was established in 2000. Membrane-bound NTPDases are either located on the cell surface or membranes of intracellular organelles. Soluble NTPDases exist in the cytosol and may be secreted. In the last 15 years, molecular cloning and functional expression have facilitated biochemical characterization of NTPDases of many organisms, culminating in the recent structural determination of the ecto-domain of a mammalian cell surface NTPDase and a bacterial NTPDase. The first goal of this review is to summarize the biochemical, mutagenesis, and structural studies of the NTPDases. Because of their ability in hydrolyzing extracellular nucleotides, the mammalian cell surface NTPDases (the ecto-NTPDases) which regulate purinergic signaling have received the most attention. Less appreciated are the functions of intracellular NTPDases and NTPDases of other organisms, e.g., bacteria, parasites, Drosophila, plants, etc. The second goal of this review is to summarize recent findings which demonstrate the involvement of the NTPDases in multiple and diverse physiological processes: pathogen-host interaction, plant growth, eukaryote cell protein and lipid glycosylation, eye development, and oncogenesis.
Nucleosides and nucleotides are now considered as extracellular signalling molecules, like neurotransmitters and hormones. Hepatic cells, amongst other cells, ubiquitously express specific transmembrane receptors that transduce the physiological signals induced by extracellular nucleosides and nucleotides, as well as various cell surface enzymes that regulate the levels of these mediators in the extracellular medium. Here, we cover various aspects of the signalling pathways initiated by extracellular nucleosides and nucleotides in the liver, and discuss their overall impact on hepatic physiology.
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.
We previously demonstrated that when platelets are in motion and in proximity to endothelial cells, they become unresponsive to agonists (Marcus, A.J., L.B. Safier, K.A. Hajjar, H.L. Ullman, N. Islam, M.J. Broekman, and A.M. Eiroa. 1991. J. Clin. Invest. 88:1690-1696). This inhibition is due to an ecto-ADPase on the surface of endothelial cells which metabolizes ADP released from activated platelets, resulting in blockade of the aggregation response. Human umbilical vein endothelial cells (HUVEC) ADPase was biochemically classified as an E-type ATP-diphosphohydrolase. The endothelial ecto-ADPase is herein identified as CD39, a molecule originally characterized as a lymphoid surface antigen. All HUVEC ecto-ADPase activity was immunoprecipitated by monoclonal antibodies to CD39. Surface localization of HUVEC CD39 was established by confocal microscopy and flow cytometric analyses. Transfection of COS cells with human CD39 resulted in both ecto-ADPase activity as well as surface expression of CD39. PCR analyses of cDNA obtained from HUVEC mRNA and recombinant human CD39 revealed products of the same size, and of identical sequence. Northern blot analyses demonstrated that HUVEC express the same sized transcripts for CD39 as MP-1 cells (from which CD39 was originally cloned). We established the role of CD39 as a prime endothelial thromboregulator by demonstrating that CD39-transfected COS cells acquired the ability to inhibit ADP-induced aggregation in platelet-rich plasma. The identification of HUVEC ADPase/CD39 as a constitutively expressed potent inhibitor of platelet reactivity offers new prospects for antithrombotic therapeusis.
Excessive platelet accumulation and recruitment, leading to vessel occlusion at sites of vascular injury, present major therapeutic challenges in cardiovascular medicine. Endothelial cell CD39, an ecto-enzyme with ADPase and ATPase activities, rapidly metabolizes ATP and ADP released from activated platelets, thereby abolishing recruitment. Therefore, a soluble form of CD39, retaining nucleotidase activities, would constitute a novel antithrombotic agent. We designed a recombinant, soluble form of human CD39, and isolated it from conditioned media from transiently transfected COS-1 cells and from stably transfected Chinese hamster ovary (CHO) cells. Conditioned medium from CHO cells grown under serum-free conditions was subjected to anti-CD39 immunoaffinity column chromatography, yielding a single approximately 66-kD protein with ATPase and ADPase activities. Purified soluble CD39 blocked ADP-induced platelet aggregation in vitro, and inhibited collagen-induced platelet reactivity. Kinetic analyses indicated that, while soluble CD39 had a Km for ADP of 5.9 microM and for ATP of 2.1 microM, the specificity constant kcat/Km was the same for both substrates. Intravenously administered soluble CD39 remained active in mice for an extended period of time, with an elimination phase half-life of almost 2 d. The data indicate that soluble CD39 is a potential therapeutic agent for inhibition of platelet-mediated thrombotic diatheses.
Studies on murine B lymphocytes showed that Bruton's tyrosine kinase mediates signal transduction induced via CD38, a nonlineage-restricted 45-kD ectoenzyme. This signaling is defective in B cells from X-linked immunodeficient mice affected with the analogue of human X-linked agammaglobulinemia (XLA). We performed a structural and functional analysis of CD38 in XLA and other immunodeficiencies, using EBV-immortalized B cells derived from such patients. Membrane CD38 was not significantly different from controls in structure, epitope density, enzymatic activity, and internalization upon binding of agonistic mAbs. Meanwhile, an increased release of soluble CD38 from XLA cells was observed: immunoprecipitation from XLA culture media yielded a protein of approximately 78 kD (p78), reacting also in Western blot and displaying both enzymatic activities and a peptide map similar to membrane CD38. Soluble forms and homotypic aggregations of CD38 were documented in different cell models and by crystallographic analysis of the Aplysia ADP-ribosyl cyclase, the ancestor of human CD38. p78 might represent the product of an altered turn-over of membrane CD38, a starting point for studying its association with Bruton's tyrosine kinase and its role in XLA and other B cell immunodeficiencies.
ResearchGate has not been able to resolve any references for this publication.