Inhibitors of ectonucleotidases have paradoxical effects on synaptic transmission in the mouse cortex.

Extracellular adenosine plays prominent roles in the brain in both physiological and pathological conditions. Adenosine can be generated following the degradation of extracellular nucleotides by various types of ectonucleotidases. Several ectonucleotidases are present in the brain parenchyma: ecto-nucleotide triphosphate diphosphohydrolases 1 and 3 (NTPDase 1 and 3), ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP 1), ecto-5'-nucleotidase (eN), and tissue non-specific alkaline phosphatase (TNAP, whose function in the brain has received little attention). Here we examined, in a living brain preparation, the role of these ectonucleotidases in generating extracellular adenosine. We recorded local field potentials evoked by electrical stimulation of the lateral olfactory tract in the mouse piriform cortex in vitro. Variations in adenosine level were evaluated by measuring changes in presynaptic inhibition generated by adenosine A1 receptors (A1Rs) activation. A1R-mediated presynaptic inhibition was present endogenously and was enhanced by bath-applied AMP and ATP. We hypothesized that inhibiting ectonucleotidases would reduce extracellular adenosine concentration, which would result in a weakening of presynaptic inhibition. However, inhibiting TNAP had no effect in controlling endogenous adenosine action and no effect on presynaptic inhibition induced by bath-applied AMP. Furthermore, contrary to our expectation, inhibiting TNAP reinforced, rather than reduced, presynaptic inhibition induced by bath-applied ATP. Similarly, inhibition of NTPDase 1 and 3, NPP1, and eN induced stronger, rather than weaker, presynaptic inhibition, both in endogenous condition and with bath-applied ATP and AMP. Consequently, attempts to suppress the functions of extracellular adenosine by blocking its extracellular synthesis in living brain tissue could have functional impacts opposite to those anticipated.

Protective effect of CD73 inhibitor α, ß-methylene ADP against amyloid-ß-induced cognitive impairment by inhibiting adenosine production in hippocampus

BACKGROUND: Alzheimer's disease (AD) is a chronic, progressive neurodegenerative disease. Recent studies have reported the close association between cognitive function in AD and purinergic receptors in the central nervous system. In the current study, we investigated the effect of CD73 inhibitor α, ß-methylene ADP (APCP) on cognitive impairment of AD in mice, and to explore the potential underlying mechanisms. RESULTS: We found that acute administration of Aß1­42 (i.c.v.) resulted in a significant increase in adenosine release by using microdialysis study. Chronic administration of APCP (10, 30 mg/kg) for 20 d obviously mitigated the spatial working memory impairment of Aß1­42-treated mice in both Morris water maze (MWM) test and Y-maze test. In addition, the extracellular adenosine production in the hippocampus was inhibited by APCP in Aß-treated mice. Further analyses indicated expression of acetyltransferase (ChAT) in hippocampus of mice of was significantly reduced, while acetylcholinesterase (AChE) expression increased, which compared to model group. We observed that APCP did not significantly alter the NLRP3 inflammasome activity in hippocampus, indicating that anti-central inflammation seems not to be involved in APCP effect. CONCLUSIONS: In conclusion, we report for the first time that inhibition of CD73 by APCP was able to protect against memory loss induced by Aß1­42 in mice, which may be due to the decrease of CD73-driven adenosine production in hippocampus. Enhancement of central cholinergic function of the central nervous system may also be involved in the effects of APCP.

Ectonucleotidases in Blood Malignancies: A Tale of Surface Markers and Therapeutic Targets.

Leukemia develops as the result of intrinsic features of the transformed cell, such as gene mutations and derived oncogenic signaling, and extrinsic factors, such as a tumor-friendly, immunosuppressed microenvironment, predominantly in the lymph nodes and the bone marrow. There, high extracellular levels of nucleotides, mainly NAD+ and ATP, are catabolized by different ectonucleotidases, which can be divided in two families according to substrate specificity: on one side those that metabolize NAD+, including CD38, CD157, and CD203a; on the other, those that convert ATP, namely CD39 (and other ENTPDases) and CD73. They generate products that modulate intracellular calcium levels and that activate purinergic receptors. They can also converge on adenosine generation with profound effects, both on leukemic cells, enhancing chemoresistance and homing, and on non-malignant immune cells, polarizing them toward tolerance. This review will first provide an overview of ectonucleotidases expression within the immune system, in physiological and pathological conditions. We will then focus on different hematological malignancies, discussing their role as disease markers and possibly pathogenic agents. Lastly, we will describe current efforts aimed at therapeutic targeting of this family of enzymes.

3'nucleotidase/nuclease in protozoan parasites: Molecular and biochemical properties and physiological roles.

3'-nucleotidase/nuclease (3'NT/NU) is a bi-functional enzyme that is able to hydrolyze 3'-monophosphorylated nucleotides and nucleic acids. This review summarizes the major molecular and biochemical properties of this enzyme in different trypanosomatid species. Sequence analysis of the gene encoding 3'NT/NU in Leishmania and Crithidia species showed that the protein possesses five highly conserved regions that are characteristic of members of the class I nuclease family. 3'NT/NU presents a molecular weight of approximately 40 kDa, which is conserved among the studied species. Throughout the review, we discuss inhibitors and substrate specificity, relating them to the putative structure of the enzyme. Finally, we present the major biological roles performed by 3'NT/NU. The involvement of 3'NT/NU in the purine salvage pathway was confirmed by the increase of activity and expression of the enzyme when the parasites were submitted to purine starvation. The generation of extracellular adenosine is also important to the modulation of the host immune response. Interaction assays involving Leishmania parasites and macrophages indicated that 3'-nucleotidase activity increases the association index between them. Recently, it was shown that 3'NT/NU plays a role in parasite escape from neutrophil extracellular traps, one of the first mechanisms of the host immune system for preventing infection.

Inhibitory effects promoted by 5'-nucleotides on the ecto-3'-nucleotidase activity of Leishmania amazonensis.

The protozoan parasite Leishmania amazonensis is the etiological agent of cutaneous leishmaniasis. During its life cycle, the flagellated metacyclic promastigote forms are transmitted to vertebrate hosts by sandfly bites, and they develop into amastigotes inside macrophages, where they multiply. L. amazonensis possesses a bifunctional enzyme, called 3'-nucleotidase/nuclease (3'NT/NU), which is able to hydrolyze extracellular 3'-monophosphorylated nucleosides and nucleic acids. 3'NT/NU plays an important role in the generation of extracellular adenosine and has been described as a key enzyme in the acquisition of purines by trypanosomatids. Furthermore, it has been observed that 3'NT/NU also plays a valuable role in the establishment of parasitic infection. In this context, this study aimed to investigate the modulation of the 3'-nucleotidase (3'NT) activity of L. amazonensis by several nucleotides. It was observed that 3'NT activity is inhibited by micromolar concentrations of guanosine and guanine nucleotides. The inhibition promoted by 5'-GMP on the 3'NT activity of L. amazonensis is reversible and uncompetitive because the addition of the inhibitor decreased the kinetic parameters Km and Vmax. Finally, we found that the addition of 5'-GMP is able to reverse the stimulation promoted by 3'-AMP in a macrophage-parasite interaction assay. The determination of compounds that can inhibit the 3'NT activity of Leishmania is very important because this enzyme does not occur in mammals, making it a potential therapeutic target.

Postsynaptic Potentiation in Mouse Motor Synapses Induced by ATP Accumulation in Synaptic Cleft.

In mouse motor synapses, a non-selective purinoceptor antagonist suramin increased the quantum content of endplate potentials (EPP) without changing the time course of synaptic potentials. An ectonucleotidase inhibitor ARL 67156 had no effect on the amplitude and quantum content of EPP and miniature endplate potentials (mEPP) evoked by single stimuli, but significantly prolonged their duration. Long-term high-frequency stimulation of the nerve in the presence of ARL 67156 persistently increased the amplitude and duration of EPP during the train of impulses, but did not change their quantum content. ATP-γ-S, a non-hydrolyzed ATP analogue, significantly increased the amplitudes and prolonged the rising and falling phases of EPP and mEPP. The ATP-induced postsynaptic potentiation in neuromuscular transmission can result from the increase in ATP content and its longer presence in the synaptic cleft.

Backbone resonance assignments of human cytosolic dNT-1 nucleotidase.

Cytosolic dNT-1 nucleotidase plays a key role in the homeostasis of pyrimidine deoxyribonucleotides in mammalian cells. The enzyme is responsible for the dephosphorylation of physiological substrates as well as nucleoside analogues that are used in antiviral and anticancer therapies, therefore selective inhibition of the dNT-1 nucleotidase activity may lead to an increase in efficacy of this type of therapeutic compounds. Here, we report the backbone ¹H, ¹³C and ¹5N assignments for the 47 kDa dNT-1 dimer, which will be used for structural characterisation of dNT-1 complexes with small molecule inhibitors obtained through modification of pyrimidine nucleotide scaffolds or optimisation of successful binders obtained from the screening of fragment libraries.

Inhibition of extracellular nucleotides hydrolysis intensifies the allergic bronchospasm. A novel protective role of ectonucleotidases.

BACKGROUND: Nucleotides released to the extracellular space stimulate purinergic receptors, and their effects are modulated by ectonucleotidases. The role of ATP in the allergic bronchospasm has been scantly studied. METHODS: We used several techniques (plethysmography, organ baths, confocal microscopy, RT-PCR, ATP measurement) to explore the role of nucleotides and ectonucleotidases in the allergic bronchospasm in guinea pigs. RESULTS: While allergenic challenge with a low-dose ovalbumin (OVA) only produced a small bronchospasm (~2-fold the basal lung resistance), previous inhibition of ectonucleotidases by ARL-67156 greatly intensified this response (~11-fold the basal lung resistance, with 44% mortality). Bronchoalveolar lavage fluid obtained during this bronchospasm contained increased ATP concentration. This potentiation was abolished by antagonism of purinergic receptors (suramin+RB2) or TXA2 receptor (SQ29548), or by intratracheal apyrase. In tracheal rings and lung parenchyma strips, OVA caused a concentration-dependent contraction. Suramin+RB2 or levamisole produced a significant rightward displacement of this response, and ARL-67156 did not modify it. Platelets stimulated with OVA released ATP. Confocal images of nonsensitized tracheas showed slight fluorescence for P2Y6 receptors in epithelium and none for P2Y4 . Sensitized animals showed strong fluorescence to both receptors and to alkaline phosphatase in the airway epithelium. This correlated with a large increment in mRNA for P2Y4 and P2Y6 receptors in sensitized animals. CONCLUSIONS: Nucleotides greatly potentiate the allergic bronchospasm when ectonucleotidases activity is diminished, and this effect is probably favored by the upregulation of P2Y4 and P2Y6 receptors in airway epithelium during sensitization. These results prompt for further research on these mechanisms in human asthma.

Ectonucleotidases and nucleotide/nucleoside transporters as pharmacological targets for neurological disorders.

Extracellular nucleotide and nucleoside are signaling molecules with a wide range of actions in the central nervous system (CNS). Extracellular ATP is released by several mechanisms involving ATP binding cassette transporters, hemichannels, P2X7 receptors, or volume-sensitive chloride channels. The levels of ATP and its hydrolysis product, adenosine, in the synaptic cleft are controlled by a complex cascade of cell surface-located enzymes collectively known as ectonucleotidases. There are four major families of ectonucleotidases: ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases), ecto-nucleotide pyrophosphatase/phosphodiesterases (E-NPPs), alkaline phosphatases, and ecto-5'- nucleotidase. Besides the production of adenosine through nucleotide hydrolysis, this neuromodulator can be released as adenosine per se by equilibrative and/or concentrative nucleoside transporters. In this review, the involvement of nucleotide/nucleoside transporters and ectonucleotidases in the pathophysiology of brain disorders is discussed. The identification of compounds able to modulate the activity of these players in purinergic neurotransmission and their implications in neurological disorders as potential targets for drug discovery is also highlighted.

Leishmania amazonensis: inhibition of 3'-nucleotidase activity by Cu2+ ions.

Free Cu(2+) is toxic due to the capacity of free copper ions to catalyze the production of reactive oxygen species (ROS) that can modify the structure and/or function of biomolecules. In addition, non-specific binding to enzymes, which modifies their catalytic activities, can occur. In this work, the mechanisms underlying the ability of copper to inhibit 3'-nucleotidase from Leishmania amazonensis (La3'-nucleotidase) were investigated. To that end, La3'-nucleotidase activity was assayed with CuCl(2) in the presence of ascorbate or hydrogen peroxide to discriminate non-specific binding effects from pro-oxidant effects of copper. Copper inhibitory effects were greater at more acidic pH than at alkaline pH. The addition of enzyme substrate, adenosine 3'-monophosphate (3'AMP), prevented the inhibition of enzyme activity by copper. Thiol-containing compounds were able to protect the enzyme activity against inhibition due to copper. The specific copper chelating agent bathocuproine sulphonate (BCS) restored enzyme activity after pre-treatment of the enzyme with copper. La3'-nucleotidase activity was found to be resistant to ROS generated during oxidation reactions of ascorbate and hydrogen peroxide catalyzed by copper. Our results suggest that Cu(2+) ions exert their inhibitory effects by binding to specific motifs of the 3'-nucleotidase protein and that the enzyme appears to be extremely resistant to ROS.

Enzymatic properties of Staphylococcus aureus adenosine synthase (AdsA).

BACKGROUND: Staphylococcus aureus is a human pathogen that produces extracellular adenosine to evade clearance by the host immune system, an activity attributed to the 5'-nucleotidase activity of adenosine synthase (AdsA). In mammals, conversion of adenosine triphosphate to adenosine is catalyzed in a two-step process: ecto-nucleoside triphosphate diphosphohydrolases (ecto-NTDPases) hydrolyze ATP and ADP to AMP, whereas 5'-nucleotidases hydrolyze AMP to adenosine. NTPDases harbor apyrase conserved regions (ACRs) that are critical for activity. RESULTS: NTPDase ACR motifs are absent in AdsA, yet we report here that recombinant AdsA hydrolyzes ADP and ATP in addition to AMP. Competition assays suggest that hydrolysis occurs following binding of all three substrates at a unique site. Alanine substitution of two amino acids, aspartic acid 127 and histidine 196 within the 5'-nucleotidase signature sequence, leads to reduced AMP or ADP hydrolysis but does not affect the binding of these substrates. CONCLUSION: Collectively, these results provide insight into the unique ability of AdsA to produce adenosine through the consecutive hydrolysis of ATP, ADP and AMP, thereby endowing S. aureus with the ability to modulate host immune responses.

The effect of curcumin in the ectonucleotidases and acetylcholinesterase activities in synaptosomes from the cerebral cortex of cigarette smoke-exposed rats.

With the evidence that curcumin may be a potent neuroprotective agent and that cigarette smoke is associated with a decline in the cognitive performance as our bases, we investigated the activities of Ecto-Nucleoside Triphosphate Diphosphohydrolase (NTPDase), 5'-nucleotidase and acetylcholinesterase (AChE) in cerebral cortex synaptosomes from cigarette smoke-exposed rats treated with curcumin (Cur). The experimental procedures entailed two sets of experiments. In the first set, the groups were vehicle, Cur 12·5, 25 and 50 mg·kg(-1) ; those in the second set were vehicle, smoke, smoke and Cur 12·5, 25 and 50 mg·kg(-1) . Curcumin prevented the increased NTPDase, 5'-nucleotidase and AChE activities caused by smoke exposure. We suggest that treatment with Cur was protective because the decrease of ATP and acetylcholine (ACh) concentrations is responsible for cognitive impairment, and both ATP and ACh have key roles in neurotransmission.

Impact of ectoenzymes on p2 and p1 receptor signaling.

P2 receptors that are activated by extracellular nucleotides (e.g., ATP, ADP, UTP, UDP, Ap(n)A) and P1 receptors activated by adenosine control a diversity of biological processes. The activation of these receptors is tightly regulated by ectoenzymes that metabolize their ligands. This review presents these enzymes as well as their roles in the regulation of P2 and P1 receptor activation. We focus specifically on the role of ectoenzymes in processes of our interest, that is, inflammation, vascular tone, and neurotransmission. An update on the development of ectonucleotidase inhibitors is also presented.

Studies of the extracellular ATP-adenosine pathway in human urinary tract epithelial cells.

AIMS: Extracellular ATP may be metabolized to AMP and adenosine by the ectonucleotidases CD39 and CD73 and, in this study, we characterized the pathways for adenosine formation in human urinary tract epithelial cells. METHODS: Bladder (RT4) and kidney (A498) epithelial cells were grown in cell culture and the expression of CD39 and CD73 was investigated by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. High-performance liquid chromatography was used to determine adenosine formation in cell medium. RESULTS: RT-PCR and immunohistochemistry revealed a high CD73 and a low CD39 expression in human urinary tract epithelial cells, whereas neutrophils had a higher CD39 than CD73 expression. Adenosine was produced when the cells were exposed to 5'-AMP (substrate for CD73), but not when exposed to 5'-ATP (substrate for CD39). A pronounced inhibition of 5'-AMP-induced adenosine formation by the CD73 inhibitor AMP-CP confirmed the involvement of CD73. Adenosine production from 5'-ATP was slightly increased (p < 0.05) when epithelial cells were cocultured with neutrophils. CONCLUSIONS: The data demonstrate that adenosine formation from extracellular ATP is negligible in urinary tract epithelial cells due to low CD39 expression in this cell type. However, the epithelial cells express CD73 and are able to convert extracellular AMP to adenosine.

Steroid hormones alter AMP hydrolysis in intact trophozoites of Trichomonas vaginalis.

Trichomonas vaginalis infection may be influenced by the vaginal concentrations of estrogens. We have investigated the effects of 17beta-estradiol and dehydroepiandrosterone sulfate (DHEAS) on the ecto-5'-nucleotidase activity in fresh clinical (VP60) and in long-term-grown (30236 ATCC) isolates of T. vaginalis. In vitro exposure to DHEAS and 17beta-estradiol did not induce any changes in adenosine monophosphate (AMP) hydrolysis in these isolates. The treatment of parasites in the presence of DHEAS (0.01-1.0 microM) for 2 h inhibited AMP hydrolysis in VP60 isolate, whereas there were no significant changes in nucleotide hydrolysis in the presence of 17beta-estradiol. DHEAS and 17beta-estradiol (0.01-1.0 microM) for 2 h inhibited AMP hydrolysis in 30236 isolate. The 12 treatment with 0.1 microM DHEAS inhibited AMP hydrolysis, whereas 17beta-estradiol did not alter the nucleotide hydrolysis in VP60 isolate. Our findings have shown that the complex effect of steroid hormones and their receptors on T. vaginalis may promote changes in ecto-5'-nucleotidase activity during exposure to these hormones.

Characterization of a functional NTPDase in the endoplasmic reticulum of rat submandibular salivary gland.

Nucleotidase activity and Ca-uptake were characterized in endoplasmic reticulum (ER) enriched rat submandibular gland (SMG) microsomal preparations. (i) Ca-uptake had characteristics of an ER Ca-ATPase. (ii) Nucleotidase activity was equally stimulated by calcium, magnesium and manganese, but with different Km values. (iii) Specific inhibitors of P-type Ca-ATPases were ineffective on nucleotidase activity, demonstrating that this activity was not related to calcium uptake and did not correspond to classical Ca(2+) pumps. (iv) ATP and UTP were more efficient substrates, whereas ADP and UDP were hydrolyzed at significantly slower rate. (v) Nucleotidase activity was sensitive to mild detergent solubilization and insensitive to ionophore addition. (vi) Nucleotidase activity was strongly inhibited by suramin, a nucleoside triphosphate diphosphohydrolase (NTPDase) inhibitor. (vii) Nucleotidase activity exponentially diminished as function of time. All these observations are consistent with a NTPDase identity. The presence of a NTPDase was demonstrated by immunohistochemistry in rat SMG. Immunoreactivity was stronger in ductal cells than in mucous and serous acini. Although this enzyme was observed in the plasma membrane, colocalization with the ER marker calnexin revealed a specific subcellular localization in this organelle of all three types of cell. The putative function of this NTPDase activity in salivary glands is discussed.

ATP release and extracellular nucleotidase activity in erythrocytes and coronary circulation of rainbow trout.

The present study tested the hypothesis that rainbow trout erythrocytes release ATP upon deoxygenation, a mechanism that enables mammalian erythrocytes to produce local vasodilation. We also investigated ATP release and ectonucleotidase activity in the coronary circulation of the isolated trout heart. Erythrocytes suspended in an albumin-containing saline and equilibrated at physiological Pco2 showed negligible hemolysis (<0.1%), and notably they released small amounts of ATP. The elevation of extracellular [ATP] was higher in the presence of the ectonucleotidase inhibitor ARL 67156 than in its absence, revealing the presence of ectonucleotidase activity. The induction of either a slow (minutes) or a fast (seconds) decrease in hemoglobin O2 saturation did not lead to additional ATP release. An elevation of Pco(2) was also without influence on erythrocyte ATP release. In the saline-perfused coronary circulation, [ATP] increased as the perfusate moved through the vessels in the presence of ARL 67156. When ATP was added to the inflowing saline, most ATP disappeared during passage of the coronary bed when ARL 67156 was absent but not when it was present. We conclude that rainbow trout erythrocytes and vasculature possess the key elements for ATP signaling, i.e. cellular ATP release and balanced ATP degradation by ectonucleotidases, but that erythrocyte ATP release is not influenced by oxygenation degree. The latter is suggested to be related to the lack of a deoxygenation-dependent interaction of trout hemoglobin with the cytoplasmic domain of band 3.

A role for a lithium-inhibited Golgi nucleotidase in skeletal development and sulfation.

Sulfation is an important biological process that modulates the function of numerous molecules. It is directly mediated by cytosolic and Golgi sulfotransferases, which use 3'-phosphoadenosine 5'-phosphosulfate to produce sulfated acceptors and 3'-phosphoadenosine 5'-phosphate (PAP). Here, we identify a Golgi-resident PAP 3'-phosphatase (gPAPP) and demonstrate that its activity is potently inhibited by lithium in vitro. The inactivation of gPAPP in mice led to neonatal lethality, lung abnormalities resembling atelectasis, and dwarfism characterized by aberrant cartilage morphology. The phenotypic similarities of gPAPP mutant mice to chondrodysplastic models harboring mutations within components of the sulfation pathway lead to the discovery of undersulfated chondroitin in the absence of functional enzyme. Additionally, we observed loss of gPAPP leads to perturbations in the levels of heparan sulfate species in lung tissue and whole embryos. Our data are consistent with a model that clearance of the nucleotide product of sulfotransferases within the Golgi plays an important role in glycosaminoglycan sulfation, provide a unique genetic basis for chondrodysplasia, and define a function for gPAPP in the formation of skeletal elements derived through endochondral ossification.

Specificity of the ecto-ATPase inhibitor ARL 67156 on human and mouse ectonucleotidases.

BACKGROUND AND PURPOSE: ARL 67156, 6-N,N-Diethyl-D-beta-gamma-dibromomethylene adenosine triphosphate, originally named FPL 67156, is the only commercially available inhibitor of ecto-ATPases. Since the first report on this molecule, various ectonucleotidases responsible for the hydrolysis of ATP at the cell surface have been cloned and characterized. In this work, we identified the ectonucleotidases inhibited by ARL 67156. EXPERIMENTAL APPROACH: The effect of ARL 67156 on recombinant NTPDase1, 2, 3 & 8 (mouse and human), NPP1, NPP3 and ecto-5'-nucleotidase (human) have been evaluated. The inhibition of the activity of NTPDases (using the following substrates: ATP, ADP, UTP), NPPs (pnp-TMP, Ap(3)A) and ecto-5'-nucleotidase (AMP) was measured by colorimetric or HPLC assays. KEY RESULTS: ARL 67156 was a weak competitive inhibitor of human NTPDase1, NTPDase3 and NPP1 with K(i) of 11+/-3, 18+/-4 and 12+/-3 microM, respectively. At concentrations used in the literature (50-100 microM), ARL 67156 partially but significantly inhibited the mouse and human forms of these enzymes. NTPDase2, NTPDase8, NPP3 and ecto-5'-nucleotidase activities were less affected. Importantly, ARL 67156 was not hydrolysed by either human NTPDase1, 2, 3, 8, NPP1 or NPP3. CONCLUSIONS AND IMPLICATIONS: In cell environments where NTPDase1, NTPDase3, NPP1 or mouse NTPDase8 are present, ARL 67156 would prolong the effect of endogenously released ATP on P2 receptors. However, it does not block any ectonucleotidases efficiently when high concentrations of substrates are present, such as in biochemical, pharmacological or P2X(7) assays. In addition, ARL 67156 is not an effective inhibitor of NTPDase2, human NTPDase8, NPP3 and ecto-5'-nucleotidase.

Extracellular ATP induces assembly and activation of the myosin light chain phosphatase complex in endothelial cells.

OBJECTIVES: Extracellular ATP stabilizes the endothelial barrier and inactivates the contractile machinery of endothelial cells. This inactivation relies on dephosphorylation of the regulatory myosin light chain (MLC) due to an activation of the MLC phosphatase (MLCP). To date, activation and function of MLCP in endothelial cells are only partially understood. METHODS: Here, the mechanism of extracellular ATP-mediated activation of MLCP was analyzed in human endothelial cells from umbilical veins. Cells were transfected with the endogenous protein phosphatase 1 (PP1)-specific inhibitor-2 (I-2). RESULTS: Overexpression of I-2 led to inhibition of PP1 activity and abrogation of the ATP-induced dephosphorylation of MLC. This indicates that the PP1 catalytic subunit is the principal phosphatase catalyzing the MLC dephosphorylation induced by extracellular ATP. As demonstrated by immunoprecipitation analysis, extracellular ATP recruits the PP1delta catalytic subunit and the myosin phosphatase targeting subunit (MYPT1) to form a complex. ATP stimulated dephosphorylation of MYPT1 at the inhibitory phosphorylation sites threonine 850 and 696. However, extracellular ATP failed to stimulate MYPT1 dephosphorylation in I-2-overexpressing cells. CONCLUSIONS: The present study shows for the first time that, in endothelial cells, extracellular ATP causes activation of MLCP through recruitment of PP1delta and MYPT1 into a MLCP holoenzyme complex and PP1-mediated reduction of the inhibitory phosphorylation of MYPT1.