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1.
Kukulski F Lévesque SA Lavoie EG Lecka J Bigonnesse F Knowles AF Robson SC Kirley TL Sévigny J 《Purinergic signalling》2005,1(2):193-204
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 Km values in the micromolar range, indicating that they should terminate the effects exerted by these nucleotide agonists at P2X1–7 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 7.0–8.5. 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. 相似文献
2.
《Journal of structural biology》2014,185(3):336-341
In many vertebrate tissues CD39-like ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) act in concert with ecto-5′-nucleotidase (e5NT, CD73) to convert extracellular ATP to adenosine. Extracellular ATP is a cytotoxic, pro-inflammatory signalling molecule whereas its product adenosine constitutes a universal and potent immune suppressor. Interference with these ectonucleotidases by use of small molecule inhibitors or inhibitory antibodies appears to be an effective strategy to enhance anti-tumour immunity and suppress neoangiogenesis. Here we present the first crystal structures of an NTPDase catalytic ectodomain in complex with the Reactive Blue 2 (RB2)-derived inhibitor PSB-071. In both of the two crystal forms presented the inhibitor binds as a sandwich of two molecules at the nucleoside binding site. One of the molecules is well defined in its orientation. Specific hydrogen bonds are formed between the sulfonyl group and the nucleoside binding loop. The methylphenyl side chain functionality that improved NTPDase2-specificity is sandwiched between R245 and R394, the latter of which is exclusively found in NTPDase2. The second molecule exhibits great in-plane rotational freedom and could not be modelled in a specific orientation. In addition to this structural insight into NTPDase inhibition, the observation of the putative membrane interaction loop (MIL) in two different conformations related by a 10° rotation identifies the MIL as a dynamic section of NTPDases that is potentially involved in regulation of catalysis. 相似文献
3.
Lavoie EG Gulbransen BD Martín-Satué M Aliagas E Sharkey KA Sévigny J 《American journal of physiology. Gastrointestinal and liver physiology》2011,300(4):G608-G620
Extracellular nucleotides and adenosine are biologically active molecules that bind members of the P2 and P1 receptor families, respectively. In the digestive system, these receptors modulate various functions, including salivary, gastric, and intestinal epithelial secretion and enteric neurotransmission. The availability of P1 and P2 ligands is modulated by ectonucleotidases, enzymes that hydrolyze extracellular nucleotides into nucleosides. Nucleoside triphosphate diphosphohydrolases (NTPDases) and ecto-5'-nucleotidase are the dominant ectonucleotidases at physiological pH. While there is some information about the localization of ecto-5'-nucleotidase and NTPDase1 and -2, the distribution of NTPDase3 in the digestive system is unknown. We examined the localization of these ectonucleotidases, with a focus on NTPDase3, in the gastrointestinal tract and salivary glands. NTPDase1, -2, and -3 are responsible for ecto-ATPase activity in these tissues. Semiquantitative RT-PCR, immunohistochemistry, and in situ enzyme activity revealed the presence of NTPDase3 in some epithelial cells in serous acini of salivary glands and mucous acini and duct cells of sublingual salivary glands, in cells from the stratified esophageal and forestomach epithelia, and in some enteroendocrine cells of the gastric antrum. Interestingly, NTPDase2 and ecto-5'-nucleotidase are coexpressed with NTPDase3 in salivary gland cells and stratified epithelia. In the colon, neurons express NTPDase3 and glial cells express NTPDase2. Ca(2+) imaging experiments demonstrate that NTPDases regulate P2 receptor ligand availability in the enteric nervous system. In summary, the specific localization of NTPDase3 in the digestive system suggests functional roles of the enzyme, in association with NTPDase2 and ecto-5'-nucleotidase, in epithelial functions such as secretion and in enteric neurotransmission. 相似文献
4.
Mireia Martín-Satué Elise G. Lavoie Michel Fausther Joanna Lecka Elisabet Aliagas Filip Kukulski Jean Sévigny 《Histochemistry and cell biology》2010,133(6):659-668
Extracellular ATP and its hydrolysis product adenosine modulate various reproductive functions such as those requiring contraction,
steroidogenesis, and maintenance of fluid composition. Interestingly, adenosine might act as a key capacitative effector for
mammalian spermatozoa to acquire the capacity for fertilisation. Extracellular nucleotide levels are affected by cell surface
ectonucleotidases, amongst which the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family regroups the most abundant
and effective enzymes to hydrolyse ATP and ADP to AMP in physiological conditions. In the male reproductive tract three members
of this family have been indentified: NTPDase1, NTPDase2 and NTPDase3 (Martín-Satué et al. in Histochem Cell Biol 131:615–628,
2009). The purpose of the present study was to characterize in the male reproductive tract the expression profile of the main
enzyme responsible for the generation of adenosine from AMP, namely the ecto-5′-nucleotidase (CD73). The enzyme was identified
by immunological techniques and by in situ enzymatic assays, including inhibition experiments with α,β-methylene-ADP, a specific
CD73 inhibitor. High levels of ecto-5′-nucleotidase were detected in testes in association with both germinal and somatic
cells, in smooth muscle cells throughout the tract, in secretory epithelia from exocrine glands, and remarkably, in principal
cells of epididymis, where co-localization with NTPDase3 was found. The relevance of this co-expression on nucleotide hydrolysis
in these cells directly involved in the control of sperm fluid composition was addressed biochemically. This study suggests
close regulation of extracellular nucleoside and nucleotide levels in the genital tract by ecto-5′-nucleotidase that, in concurrence
with NTPDases, may impact male fertility. 相似文献
5.
Fausther M Lecka J Kukulski F Lévesque SA Pelletier J Zimmermann H Dranoff JA Sévigny J 《American journal of physiology. Gastrointestinal and liver physiology》2007,292(3):G785-G795
Extracellular nucleotides regulate critical liver functions via the activation of specific transmembrane receptors. The hepatic levels of extracellular nucleotides, and therefore the related downstream signaling cascades, are modulated by cell-surface enzymes called ectonucleotidases, including nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39), NTPDase2/CD39L1, and ecto-5'-nucleotidase/CD73. The goal of this study was to determine the molecular identity of the canalicular ecto-ATPase/ATPDase that we hypothesized to correspond to the recently cloned NTPDase8. Human and rat NTPDase8 cDNAs were cloned, and the genes were located on chromosome loci 9q34 and 3p13, respectively. The recombinant proteins, expressed in COS-7 and HEK293T cells, were biochemically characterized. NTPDase8 was also purified from rat liver by Triton X-100 solubilization, followed by DEAE, Affigel Blue, and concanavalin A chromatographies. Importantly, NTPDase8 was responsible for the major ectonucleotidase activity in liver. The ion requirement, apparent K(m) values, nucleotide hydrolysis profile, and preference as well as the resistance to azide were similar for recombinant NTPDase8s and both purified rat NTPDase8 and porcine canalicular ecto-ATPase/ATPDase. The partial NH(2)-terminal amino acid sequences of all NTPDase8s share high identity with the purified liver canalicular ecto-ATPase/ATPDase. Histochemical analysis showed high ectonucleotidase activities in bile canaliculi and large blood vessels of rat liver, in agreement with the immunolocalization of NTPDase1, 2, and 8 with antibodies developed for this study. No NTPDase3 expression could be detected in liver. In conclusion, NTPDase8 is the canalicular ecto-ATPase/ATPDase and is responsible for the main hepatic NTPDase activity. The canalicular localization of this enzyme suggests its involvement in the regulation of bile secretion and/or nucleoside salvage. 相似文献
6.
Members of all four families of ectonucleotidases, namely ectonucleoside triphosphate diphosphohydrolases (NTPDases), ectonucleotide
pyrophosphatase/phosphodiesterases (NPPs), ecto-5′-nucleotidase and alkaline phosphatases, have been identified in the renal
vasculature and/or tubular structures. In rats and mice, NTPDase1, which hydrolyses ATP through to AMP, is prominent throughout
most of the renal vasculature and is also present in the thin ascending limb of Henle and medullary collecting duct. NTPDase2
and NTPDase3, which both prefer ATP over ADP as a substrate, are found in most nephron segments beyond the proximal tubule.
NPPs catalyse not only the hydrolysis of ATP and ADP, but also of diadenosine polyphosphates. NPP1 has been identified in
proximal and distal tubules of the mouse, while NPP3 is expressed in the rat glomerulus and pars recta, but not in more distal
segments. Ecto-5′-nucleotidase, which catalyses the conversion of AMP to adenosine, is found in apical membranes of rat proximal
convoluted tubule and intercalated cells of the distal nephron, as well as in the peritubular space. Finally, an alkaline
phosphatase, which can theoretically catalyse the entire hydrolysis chain from nucleoside triphosphate to nucleoside, has
been identified in apical membranes of rat proximal tubules; however, this enzyme exhibits relatively high K
m values for adenine nucleotides. Although information on renal ectonucleotidases is still incomplete, the enzymes’ varied
distribution in the vasculature and along the nephron suggests that they can profoundly influence purinoceptor activity through
the hydrolysis, and generation, of agonists of the various purinoceptor subtypes. This review provides an update on renal
ectonucleotidases and speculates on the functional significance of these enzymes in terms of glomerular and tubular physiology
and pathophysiology. 相似文献
7.
Nucleoside triphosphate diphosphohydrolases (NTPDases) are a physiologically important class of membrane-bound ectonucleotidases responsible for the regulation of extracellular levels of nucleotides. CD39 or NTPDase1 is the dominant NTPDase of the vasculature. By hydrolyzing proinflammatory ATP and platelet-activating ADP to AMP, it blocks platelet aggregation and supports blood flow. Thus, great interest exists in understanding the structure and dynamics of this prototype member of the eukaryotic NTPDase family. Here, we report the crystal structure of a variant of soluble NTPDase1 lacking a putative membrane interaction loop identified between the two lobes of the catalytic domain. ATPase and ADPase activities of this variant are determined via a newly established kinetic isothermal titration calorimetry assay and compared to that of the soluble NTPDase1 variant characterized previously. Complex structures with decavanadate and heptamolybdate show that both polyoxometallates bind electrostatically to a loop that is involved in binding of the nucleobase. In addition, a comparison of the domain orientations of the four independent proteins in the crystal asymmetric unit provides the first direct experimental evidence for a domain motion of NTPDases. An interdomain rotation angle of up to 7.4° affects the active site cleft between the two lobes of the protein. Comparison with a previously solved bacterial NTPDase structure indicates that the domains may undergo relative rotational movements of more than 20°. Our data support the idea that the influence of transmembrane helix dynamics on activity is achieved by coupling to a domain motion. 相似文献
8.
Nucleotides and nucleosides play an important role in neurodevelopment acting through specific receptors. Ectonucleotidases
are the major enzymes involved in controlling the availability of purinergic receptors ligands. ATP is co-released with several
neurotransmitters and is the most important source of extracellular adenosine by catabolism exerted by ectonucleotidases.
The main ectonucleotidases are named NTPDases (1–8) and 5′-nucleotidase. Adenosine is a powerful modulator of neurotransmitter
release. Caffeine blocks adenosine receptor activity as well as adenosine-mediated neuromodulation. Considering the susceptibility
of the immature brain to caffeine and the need for correct purinergic signaling during fetal development, we have analyzed
the effects of caffeine exposure during gestational and lactational periods on nucleotide degradation and ectonucleotidase
expression from the hippocampi of 7-, 14- and 21-days-old rats. Nucleotides hydrolysis was assessed by colorimetric determination
of inorganic phosphate released. Ectonucleotidases expression was performed by RT-PCR. ATP and ADP hydrolysis displayed parallel
age-dependent decreases in both control and caffeine-treated groups. AMP hydrolysis increased with caffeine treatment in 7-days-old
rats (75%); although there was no significant difference in AMP hydrolysis between control (non caffeine-treated) rats and
14- or 21-days caffeine-treated rats. ADP hydrolysis was not affected by caffeine treatment. Caffeine treatment in 7- and
14-days-old rats decreased ATP hydrolysis when compared to the control group (19% and 60% decrease, respectively), but 21-days-treated
rats showed an increase in ATP hydrolysis (39%). Expression levels of NTPDase 1 and 5 decreased in hippocampi of caffeine-treated
rats. The expression of 5′-nucleotidase was not affected after caffeine exposure. The changes observed in nucleotide hydrolysis
and ectonucleotidases expression could promote subtle effects on normal neural development considering the neuromodulatory
role of adenosine. 相似文献
9.
Over the last seven years our laboratory has focused on the determination of the structural aspects of nucleoside triphosphate diphosphohydrolases (NTPDases) using site-directed mutagenesis and computational comparative protein modeling to generate hypotheses and models for the hydrolytic site and enzymatic mechanism of the family of NTPDase nucleotidases. This review summarizes these studies utilizing NTPDase3 (also known as CD39L3 and HB6), an NTPDase family member that is intermediate in its characteristics between the more widely distributed and studied NTPDase1 (also known as CD39) and NTPDase2 (also known as CD39L1 and ecto-ATPase) enzymes. Relevant site-directed mutagenesis studies of other NTPDases are also discussed and compared to NTPDase3 results. It is anticipated that many of the results and conclusions reached via studies of NTPDase3 will be relevant to understanding the structure and enzymatic mechanism of all the cell-surface members of this family (NTPDase1–3, 8), and that understanding these NTPDase enzymes will aid in modulating the many varied processes under purinergic signaling control. This review also integrates the site-directed mutagenesis results with a recent 3-D structural model for the extracellular portion of NTPDases that helps explain the importance of the apyrase conserved regions (ACRs) of the NTPDases. Utilizing this model and published work from Dr Guidotti's laboratory concerning the importance and characteristics of the two transmembrane helices and their movements in response to substrate, we present a speculative cartoon model of the enzymatic mechanism of the membrane-bound NTPDases that integrates movements of the extracellular region required for catalysis with movements of the N- and C-terminal transmembrane helices that are important for control and modulation of enzyme activity. 相似文献
10.
Pathological circumstances like inflammation or ischemic insult facilitate the release of adenine nucleotides from several
types of cells. These extracellular nucleotides are rapidly converted to adenosine by ectonucleotidases, mainly ectonucleoside
triphosphate diphosphohydrolase1 (NTPDase1/CD39) and CD73. NTPDase1/CD39 can interact with caveolins, structural proteins
of signal-transducing microdomains termed caveolae. Caveolins are thought to have physiological roles in heart ageing and
cardiac diseases. The aim of this study was to investigate the expression of NTPDase1 together with caveolins in chronic human
cardiovascular diseases and elucidate their role in human heart. The HPLC analysis showed significant increase in ATPase activity
in pathological samples from patients with ischemic heart disease. Immunostaining also showed alterations in the expression
and distribution of NTPDase1. Caveolin-1 and caveolin-2 expression was much alike in control and pathological cases, while
expression of caveolin-3 was lower in pathological samples. Changes in the expression of NTPDase1 and caveolins seem to be
independent of human cardiovascular disease. 相似文献
11.
Iandiev I Wurm A Pannicke T Wiedemann P Reichenbach A Robson SC Zimmermann H Bringmann A 《Purinergic signalling》2007,3(4):423-433
Extracellular nucleotides mediate glia-to-neuron signalling in the retina and are implicated in the volume regulation of retinal
glial (Müller) cells under osmotic stress conditions. We investigated the expression and functional role of ectonucleotidases
in Müller cells of the rodent retina by cell-swelling experiments, calcium imaging, and immuno- and enzyme histochemistry.
The swelling of Müller cells under hypoosmotic stress was inhibited by activation of an autocrine purinergic signalling cascade.
This cascade is initiated by exogenous glutamate and involves the consecutive activation of P2Y1 and adenosine A1 receptors, the action of ectoadenosine 5′-triphosphate (ATP)ases, and a nucleoside-transporter-mediated
release of adenosine. Inhibition of ectoapyrases increased the ATP-evoked calcium responses in Müller cell endfeet. Müller
cells were immunoreactive for nucleoside triphosphate diphosphohydrolases (NTPDase)2 (but not NTPDase1), ecto-5′-nucleotidase,
P2Y1, and A1 receptors. Enzyme histochemistry revealed that ATP but not adenosine 5′-diphosphate (ADP) is extracellularly metabolised
in retinal slices of NTPDase1 knockout mice. NTPDase1 activity and protein is restricted to blood vessels, whereas activity
of alkaline phosphatase is essentially absent at physiological pH. The data suggest that NTPDase2 is the major ATP-degrading
ectonucleotidase of the retinal parenchyma. NTPDase2 expressed by Müller cells can be implicated in the regulation of purinergic
calcium responses and cellular volume. 相似文献
12.
Häusler SF Montalbán del Barrio I Strohschein J Anoop Chandran P Engel JB Hönig A Ossadnik M Horn E Fischer B Krockenberger M Heuer S Seida AA Junker M Kneitz H Kloor D Klotz KN Dietl J Wischhusen J 《Cancer immunology, immunotherapy : CII》2011,60(10):1405-1418
The ectonucleotidases CD39 and CD73 degrade immune stimulatory ATP to adenosine that inhibits T and NK cell responses via the A(2A) adenosine receptor (ADORA2A). This mechanism is used by regulatory T cells (T(reg)) that are associated with increased mortality in OvCA. Immunohistochemical staining of human OvCA tissue specimens revealed further aberrant expression of CD39 in 29/36 OvCA samples, whereas only 1/9 benign ovaries showed weak stromal CD39 expression. CD73 could be detected on 31/34 OvCA samples. While 8/9 benign ovaries also showed CD73 immunoreactivity, expression levels were lower than in tumour specimens. Infiltration by CD4(+) and CD8(+) T cells was enhanced in tumour specimens and significantly correlated with CD39 and CD73 levels on stromal, but not on tumour cells. In vitro, human OvCA cell lines SK-OV-3 and OaW42 as well as 11/15 ascites-derived primary OvCA cell cultures expressed both functional CD39 and CD73 leading to more efficient depletion of extracellular ATP and enhanced generation of adenosine as compared to activated T(reg). Functional assays using siRNAs against CD39 and CD73 or pharmacological inhibitors of CD39, CD73 and ADORA2A revealed that tumour-derived adenosine inhibits the proliferation of allogeneic human CD4(+) T cells in co-culture with OvCA cells as well as cytotoxic T cell priming and NK cell cytotoxicity against SK-OV3 or OAW42 cells. Thus, both the ectonucleotidases CD39 and CD73 and ADORA2A appear as possible targets for novel treatments in OvCA, which may not only affect the function of T(reg) but also relieve intrinsic immunosuppressive properties of tumour and stromal cells. 相似文献
13.
The E-NTPDase family of ectonucleotidases: Structure function relationships and pathophysiological significance 总被引:5,自引:2,他引:3
Ectonucleotidases are ectoenzymes that hydrolyze extracellular nucleotides to the respective nucleosides. Within the past decade, ectonucleotidases belonging to several enzyme families have been discovered, cloned and characterized. In this article, we specifically address the cell surface-located members of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase/CD39) family (NTPDase1,2,3, and 8). The molecular identification of individual NTPDase subtypes, genetic engineering, mutational analyses, and the generation of subtype-specific antibodies have resulted in considerable insights into enzyme structure and function. These advances also allow definition of physiological and patho-physiological implications of NTPDases in a considerable variety of tissues. Biological actions of NTPDases are a consequence (at least in part) of the regulated phosphohydrolytic activity on extracellular nucleotides and consequent effects on P2-receptor signaling. It further appears that the spatial and temporal expression of NTPDases by various cell types within the vasculature, the nervous tissues and other tissues impacts on several patho-physiological processes. Examples include acute effects on cellular metabolism, adhesion, activation and migration with other protracted impacts upon developmental responses, inclusive of cellular proliferation, differentiation and apoptosis, as seen with atherosclerosis, degenerative neurological diseases and immune rejection of transplanted organs and cells. Future clinical applications are expected to involve the development of new therapeutic strategies for transplantation and various inflammatory cardiovascular, gastrointestinal and neurological diseases. 相似文献
14.
The ectonucleoside triphosphate diphosphohydrolases (NTPDases) control extracellular nucleotide concentrations, thereby modulating many important biological responses, including blood clotting and pain perception. NTPDases1-4 are oligomeric integral membrane proteins, whereas NTPDase5 (CD39L4) and NTPDase6 (CD39L2) are soluble monomeric enzymes, making them more amenable to thorough structural and functional analyses than the membrane-bound forms. Therefore, we report here the bacterial expression, refolding, purification, and biochemical characterization of the soluble portion of human NTPDase6. Consistent with the enzyme expressed in mammalian cells, this recombinant NTPDase6 efficiently hydrolyzes GDP, IDP, and UDP (specific activity of approximately 50000 micromol mg(-1) h(-1)), with slower hydrolysis of CDP, ITP, GTP, CTP, ADP, and UTP and virtually no hydrolysis of ATP. The K(m) for GDP (130 +/- 30 microM) is similar to that determined for the soluble rat NTPDase6 expressed in mammalian cells. The secondary structure of the refolded enzyme was determined by circular dichroism to be 33% alpha-helix, 18% beta-sheet, and 49% random coil, consistent with the secondary structure predicted from the amino acid sequence of soluble NTPDase6. Four of the five cysteine residues in the soluble NTPDase6 are highly conserved among all the NTPDases, while the fifth residue is not. Mutation of this nonconserved cysteine resulted in an enzyme very similar to wild type in its enzymology and secondary structure, indicating that this cysteine exists as a free sulfhydryl and is not essential for structure or function. The disulfide pairing of the other four cysteine residues was determined as Cys(249)-Cys(280) and Cys(340)-Cys(354) by HPLC and mass spectral analysis of tryptic peptides. Due to conservation of these cysteine residues, these two disulfide bonds are likely to exist in all NTPDases. A structural model for NTPDase6, incorporating these and other findings obtained with other NTPDases, is proposed. 相似文献
15.
Differential macrophage activation alters the expression profile of NTPDase and ecto-5'-nucleotidase
Zanin RF Braganhol E Bergamin LS Campesato LF Filho AZ Moreira JC Morrone FB Sévigny J Schetinger MR de Souza Wyse AT Battastini AM 《PloS one》2012,7(2):e31205
Macrophages are key elements in the inflammatory process, whereas depending on the micro-environmental stimulation they exhibit a pro-inflammatory (classical/M1) or an anti-inflammatory/reparatory (alternative/M2) phenotype. Extracellular ATP can act as a danger signal whereas adenosine generally serves as a negative feedback mechanism to limit inflammation. The local increase in nucleotides communication is controlled by ectonucleotidases, such as members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family and ecto-5'-nucleotidase/CD73 (ecto-5'-NT). In the present work we evaluated the presence of these enzymes in resident mice M1 (macrophages stimulated with LPS), and M2 (macrophages stimulated with IL-4) macrophages. Macrophages were collected by a lavage of the mice (6-8 weeks) peritoneal cavity and treated for 24 h with IL-4 (10 ng/mL) or LPS (10 ng/mL). Nitrite concentrations were measured using the Greiss reaction. Supernatants were harvested to determine cytokines and the ATPase, ADPase and AMPase activities were determined by the malachite green method and HPLC analysis. The expression of selected surface proteins was evaluated by flow cytometry. The results reveal that M1 macrophages presented a decreased ATP and AMP hydrolysis in agreement with a decrease in NTPDase1, -3 and ecto-5'-nucleotidase expression compared to M2. In contrast, M2 macrophages showed a higher ATP and AMP hydrolysis and increased NTPDase1, -3 and ecto-5'-nucleotidase expression compared to M1 macrophages. Therefore, macrophages of the M1 phenotype lead to an accumulation of ATP while macrophages of the M2 phenotype may rapidly convert ATP to adenosine. The results also showed that P1 and P2 purinoreceptors present the same mRNA profile in both phenotypes. In addition, M2 macrophages, which have a higher ATPase activity, were less sensitive to cell death. In conclusion, these changes in ectoenzyme activities might allow macrophages to adjust the outcome of the extracellular purinergic cascade in order to fine-tune their functions during the inflammatory set. 相似文献
16.
Extracellular ATP interacts with purinergic type 2 (P2) receptors and elicits many crucial biological functions. Extracellular ATP is sequentially hydrolyzed to ADP and AMP by the actions of defined nucleotidases, such as CD39, and AMP is converted to adenosine, largely by CD73, an ecto-5′-nucleotidase. Extracellular adenosine interacts with P1 receptors and often opposes the effects of P2 receptor activation. The balance between extracellular ATP and adenosine in the blood and extracellular fluid is regulated chiefly by the activities of CD39 and CD73, which constitute the CD39-adenosinergic axis. In recent years, several studies have shown this axis to play critical roles in transport of water/sodium, tubuloglomerular feedback, renin secretion, ischemia reperfusion injury, renal fibrosis, hypertension, diabetic nephropathy, transplantation, inflammation, and macrophage transformation. Important developments include global and targeted gene knockout and/or transgenic mouse models of CD39 or CD73, biological or small molecule inhibitors, and soluble engineered ectonucleotidases to directly impact the CD39-adenosinergic axis. This review presents a comprehensive picture of the multiple roles of CD39-adenosinergic axis in renal physiology, pathophysiology, and therapeutics. Scientific advances and greater understanding of the role of this axis in the kidney, in both health and illness, will direct development of innovative therapies for renal diseases. 相似文献
17.
The protein family of ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDase family) contains multiple members that hydrolyze nucleoside 5'-triphosphates and nucleoside 5'-diphosphates with varying preference for the individual type of nucleotide. We report the cloning and functional expression of rat NTPDase3. The rat brain-derived cDNA has an open reading frame of 1590 bp encoding 529 amino acid residues, a calculated molecular mass of 59.1 kDa and predicted N- and C-terminal hydrophobic sequences. It shares 94.3% and 81.7% amino acid identity with the mouse and human NTPDase3, respectively, and is more closely related to cell surface-located than to the intracellularly located members of the enzyme family. The NTPDase3 gene is allocated to chromosome 8q32 and organized into 11 exons. Rat NTPDase3 expressed in CHO cells hydrolyzed both nucleoside triphosphates and nucleoside diphosphates with hydrolysis ratios of ATP:ADP of 5:1 and UTP:UDP of 8:1. After addition of ATP, ADP is formed as an intermediate product that is further hydrolyzed to AMP. The enzyme is preferentially activated by Ca(2+) over Mg(2+) and reveals an alkaline pH optimum. Immunocytochemistry confirmed expression of heterologously expressed NTPDase3 to the surface of CHO cells. PC12 cells express endogenous surface-located NTPDase3. An immunoblot analysis detects NTPDase3 in all rat brain regions investigated. An alignment of the secondary structure domains of actin conserved within the actin/HSP70/sugar kinase superfamily to those of all members of the NTPDase family reveals apparent similarity. It infers that NTPDases share the two-domain structure with members of this enzyme superfamily. 相似文献
18.
Cloning and characterization of mouse nucleoside triphosphate diphosphohydrolase-8 总被引:12,自引:0,他引:12
Bigonnesse F Lévesque SA Kukulski F Lecka J Robson SC Fernandes MJ Sévigny J 《Biochemistry》2004,43(18):5511-5519
A novel mammalian plasma membrane bound nucleoside triphosphate diphosphohydrolase (NTPDase), named NTPDase8, has been cloned and characterized. Analysis of cDNA reveals an open reading frame of 1491 base pairs encoding a protein of 497 amino acid residues with an estimated molecular mass of 54650 Da and a predicted isoelectric point of 5.94. In a mouse, the genomic sequence is located on chromosome 2A3 and is comprised of 10 exons. The deduced amino acid sequence reveals eight putative N-glycosylation sites, two transmembrane domains, five apyrase-conserved regions, and 20-50% amino acid identity with other mammalian NTPDases. mRNA expression was detected in liver, jejunum, and kidney. Both intact cells and crude cell lysates from COS-7 cells expressing NTPDase8 hydrolyzed P2 receptor agonists, namely, ATP, ADP, UTP, and UDP, but did not hydrolyze AMP. There was an absolute requirement for divalent cations for the catalytic activity (Ca(2+) > Mg(2+)) with an optimal pH between 5.5 and 8.0 for ATP and 6.4 for ADP hydrolysis. Kinetic parameters derived from analysis of crude cell lysates showed that the enzyme had lower apparent K(m) values for adenine nucleotides and for triphosphonucleosides (K(m,app) of 13 microM for ATP, 41 microM for ADP, 47 microM for UTP, and 171 microM for UDP). Hydrolysis of triphosphonucleosides resulted in a transient accumulation of the corresponding diphosphonucleoside, as expected from the apparent K(m) values. Enzymatic properties of NTPDase8 differ from those of other NTPDases suggesting an alternative way to modulate nucleotide levels and consequently P2 receptor activation. 相似文献
19.
Clarissa B. Haas Marianna Lovszi Pl Pacher Priscila Oliveira de Souza Julie Pelletier Rafael Olive Leite Jean Svigny Zoltn Nmeth Elizandra Braganhol Gyrgy Hask 《Purinergic signalling》2021,17(4):713
Sepsis is life-threatening organ dysfunction caused by a dysregulated inflammatory and immune response to infection. Sepsis involves the combination of exaggerated inflammation and immune suppression. During systemic infection and sepsis, the liver works as a lymphoid organ with key functions in regulating the immune response. Extracellular nucleotides are considered damage-associated molecular patterns and are involved in the control of inflammation. Their levels are finely tuned by the membrane-associated ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) enzyme family. Although previous studies have addressed the role of NTPDase1 (CD39), the role of the other extracellular NTPDases, NTPDase2, -3, and -8, in sepsis is unclear. In the present studies we identified NTPDase8 as a top downregulated gene in the liver of mice submitted to cecal ligation-induced sepsis. Immunohistochemical analysis confirmed the decrease of NTPDase8 expression at the protein level. In vitro mechanistic studies using HepG2 hepatoma cells demonstrated that IL-6 but not TNF, IL-1β, bacteria, or lipopolysaccharide are able to suppress NTPDase8 gene expression. NTPDase8, as well as NTPDase2 and NTPDase3 mRNA was downregulated, whereas NTPDase1 (CD39) mRNA was upregulated in polymorphonuclear leukocytes from both inflamed and septic patients compared to healthy controls. Although the host’s inflammatory response of polymicrobial septic NTPDase8 deficient mice was no different from that of wild-type mice, IL-6 levels in NTPDase8 deficient mice were higher than IL-6 levels in wild-type mice with pneumonia. Altogether, the present data indicate that extracellular NTPDases are differentially regulated during sepsis.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11302-021-09819-1. 相似文献
20.
CD39 modulates endothelial cell activation and apoptosis 总被引:4,自引:0,他引:4
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