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Abstract-To investigate the role of adenosine formed extracellularly in vascular homeostasis, mice with a targeted deletion of the cd73/ecto-5Ј-nucleotidase were generated. Southern blot, RT-PCR, and Western blot analysis confirmed the constitutive knockout. In vivo analysis of hemodynamic parameters revealed no significant differences in systolic blood pressure, ejection fraction, or cardiac output between strains. However, basal coronary flow measured in the isolated perfused heart was significantly lower (Ϫ14%; PϽ0.05) in the mutant. Immunohistochemistry revealed strong CD73 expression on the endothelium of conduit vessels in wild-type (WT) mice. Time to carotid artery occlusion after ferric chloride (FeCl 3 ) was significantly reduced by 20% in cd73 Ϫ/Ϫ mice (PϽ0.05). Bleeding time after tail tip resection tended to be shorter in cd73mice (Ϫ35%). In vivo platelet cAMP levels were 0.96Ϯ0.46 in WT versus 0.68Ϯ0.27 pmol/10 6 cells in cd73 Ϫ/Ϫ mice (PϽ0.05). Under in vitro conditions, platelet aggregation in response to ADP (0.05 to 10 mol/L) was undistinguishable between the two strains. In the cremaster model of ischemia-reperfusion, the increase in leukocyte attachment to endothelium was significantly higher in cd73 Ϫ/Ϫ compared with WT littermates (WT 98% versus cd73 Ϫ/Ϫ 245%; PϽ0.005). The constitutive adhesion of monocytes in ex vivo-perfused carotid arteries of WT mice was negligible but significantly increased in arteries of cd73 Ϫ/Ϫ mice (PϽ0.05). Thus, our data provide the first evidence that adenosine, extracellularly formed by CD73, can modulate coronary vascular tone, inhibit platelet activation, and play an important role in leukocyte adhesion to the vascular endothelium in vivo. Key Words: transgenic mice Ⅲ adenosine Ⅲ ecto-5Ј-nucleotidase Ⅲ vascular inflammation Ⅲ thrombosis C D73/ecto-5Ј-nucleotidase, a 70-kDa glycosylphosphatidylinositol (GPI)-anchored cell surface molecule, is expressed on the vascular endothelium and catalyzes the extracellular conversion of 5Ј-AMP to adenosine. 1,2 CD73 is the final step of the extracellular nucleotide breakdown cascade that also involves membrane-associated CD39/ATPdiphosphohydrolase. 3 The product of CD73 is adenosine, a purine nucleoside that has been implicated in many physiological and pathophysiological events. 4 There are four known G-coupled adenosine receptors: A 1 , A 2A , A 2B , and A 3 , each of which operates via different intracellular signaling mechanisms and exhibits distinct patterns of tissue distribution. 5 In human neutrophils, adenosine A 1 and A 2 receptor occupancy mediate opposing roles for adenosine in inflammation: A 1 activation is proinflammatory, whereas the A 2 receptor plays an anti-inflammatory role. 6 A 2 receptor activation inhibits the neutrophil oxidative burst, whereas the A 3 receptor inhibits neutrophil degranulation 7 and may play an important role in inflammation by inhibiting eosinophil migration. 8 Recently, deletion of the A 2A receptor in transgenic mice revealed that this receptor is critical for the limitation a...

We have previously shown that the extracellular nucleoside triphosphate-hydrolyzing enzyme NTPDase2 is highly expressed in situ by stem/progenitor cells of the two neurogenic regions of the adult murine brain: the subventricular zone (type B cells) and the dentate gyrus of the hippocampus (residual radial glia). We explored the possibility that adult multipotent neural stem cells express nucleotide receptors and investigated their functional properties in vitro. Neurospheres cultured from the adult mouse SVZ in the presence of epidermal growth factor and fibroblast growth factor 2 expressed the ecto-nucleotidases NTPDase2 and the tissue nonspecific isoform of alkaline phosphatase, hydrolyzing extracellular ATP to adenosine. ATP, ADP and, to a lesser extent, UTP evoked rapid Ca 2+ transients in neurospheres that were exclusively mediated by the metabotropic P2Y 1 and P2Y 2 nucleotide receptors. In addition, agonists of these receptors and low concentrations of adenosine augmented cell proliferation in the presence of growth factors. Neurosphere cell proliferation was attenuated after application of the P2Y 1 -receptor antagonist MRS2179 and in neurospheres from P2Y 1 -receptor knockout mice. In situ hybridization identified P2Y 1 -receptor mRNA in clusters of SVZ cells. Our results infer nucleotide receptor-mediated synergism that augments growth factor-mediated cell proliferation. Together with the in situ data, this supports the notion that extracellular nucleotides contribute to the control of adult neurogenesis.

Nucleoside triphosphate diphosphohydrolases (NTPDases) are a recently described family of ectonucleotidases that differentially hydrolyze the ␥ and ␤ phosphate residues of extracellular nucleotides. Expression of this enzymatic activity has the potential to influence nucleotide P2 receptor signaling within the vasculature. We and others have documented that NTPDase1 (CD39, 78 kd) hydrolyzes both triphosphonucleosides and diphosphonucleosides and thereby terminates platelet aggregation responses to adenosine diphosphate (ADP). In contrast, we now show that NTPDase2 (CD39L1, 75 kd), a preferential nucleoside triphosphatase, activates platelet aggregation by converting adenosine triphosphate (ATP) to ADP, the specific agonist of P2Y 1 and P2Y 12 receptors. We developed specific antibodies to murine NTPDase1 and NTPDase2 and observed that both enzymes are present in the cardiac vasculature; NTPDase1 is expressed by endothelium, endocardium, and to a lesser extent by vascular smooth muscle, while NTPDase2 is associated with the adventitia of muscularized vessels, microvascular pericytes, and other cell populations in the subendocardial space. Moreover, NTPDase2 represents a novel marker for microvascular pericytes. IntroductionNucleoside triphosphate diphosphohydrolases (NTPDases) are a family of ectonucleotidases, previously classified as E-type ATPases, ATPDases, ecto-ATPases, or ecto-apyrases. 1-3 These enzymes differentially hydrolyze the terminal ␥ and ␤ phosphate residues of nucleotides, resulting in the rapid formation of the respective diphosphonucleosides and/or monophosphonucleosides. To date, 6 members of this NTPDase family have been identified. 3-13 NTPDase1, NTPDase2, and NTPDase3 are transmembrane proteins associated with the plasma membrane with an active site facing the extracellular space. 4,5,7,9,14 These NTPDase members have been demonstrated to differ in their substrate specificity. For example, NTPDase1 (CD39 [human] or cd39 [murine]) hydrolyzes both nucleoside triphosphates and diphosphates-eg, adenosine triphosphate (ATP) and adenosine diphosphate (ADP), 4,5 -whereas NTPDase2 (CD39L1) is a preferential nucleoside triphosphatase or ATPase. 7,[14][15][16] Extracellular nucleotides in various forms, and at different concentrations, activate multiple P2 receptors: ionotropic P2X and metabotropic P2Y receptors. [17][18][19][20] While NTPDases would be anticipated to generally terminate P2 receptor agonist signaling, we have also observed that NTPDase1 may prevent receptor desensitization by the catalysis of extracellular nucleotides. 21 NTPDase1 may then also facilitate subsequent responses to "pulses" of nucleotides. In addition, NTPDases in tandem with ecto-5Ј-nucleotidase may facilitate the salvage of nucleotides by the ultimate generation of dephosphorylated forms that are taken up by cells via specific transporters. 1,22 NTPDase1 is the major ectonucleotidase at the luminal surface of blood vessels. 5,21,23,24 By converting the ADP released from activated platelets, NTPDase1 modulates platel...

The recently cloned ecto-ATPase and ecto-apyrase (ecto-ATP diphosphohydrolase) are plasma-membranebound enzymes responsible for the extracellular degradation of nucleoside 5 H -triphosphates and nucleoside 5 H -diphosphates. We expressed the rat-derived enzymes in CHO cells to compare their molecular and functional properties. Sequence-specific polyclonal antibodies differentiate between the two proteins and reveal identical molecular masses of 70±80 kDa. Both enzymes are stimulated by either Ca 2+ or Mg 2+ and reveal a broad substrate specificity towards purine and pyrimidine nucleotides. Whereas ecto-apyrase hydrolyzes nucleoside 5 H -diphosphates at a rate <20±30% lower than nucleoside-5 H -triphosphates, ecto-ATPase hydrolyzes nucleoside-5 H -diphosphates only to a marginal extent. The sensitivity of the two enzymes to the inhibitors of P2 receptors suramin, PPADS and reactive blue differs. Hydrolysis of ATP by ecto-ATPase leads to the accumulation in the medium of extracellular ADP as an intermediate product, whereas ecto-apyrase dephosphorylates ATP directly to AMP. Our results suggest that previous data describing extracellular hydrolysis of ATP by a variety of intact cellular systems with unidentified ecto-nucleotidases may be explained by the coexpression of ecto-ATPase and ecto-apyrase.Keywords: ADP; ATP; ecto-apyrase; ecto-ATPase; ecto-ATP diphosphohydrolase.A surface-located enzyme cascade catalyzes the sequential degradation of extracellular ATP to ADP, AMP and adenosine in a large variety of tissues and cell types. Until recently ecto-5 Hnucleotidase, which catalyzes the final hydrolysis step from AMP to adenosine was the only enzyme defined in molecular terms [1]. The ATP and ADP hydrolyzing ecto-nucleotidases are not inhibited by known inhibitors of intracellular ATPases such as P-type, F-type and V-type ATPases or alkaline phosphatase, suggesting that they represent a novel class of nucleotidehydrolyzing enzymes [2,3]. A major function of the extracellular enzyme chain appears to be termination of the physiological action of nucleotides released from cells. Nucleotides represent a ubiquitous class of cell-to-cell signaling substances eliciting physiological responses in every tissue. ATP, ADP, UTP and UDP exert their function via surface-located receptors, which have a tissue distribution as broad as that of the ecto-nucleotidases. Their receptor-mediated actions are involved in a large variety of physiological and pathophysiological functions [4].Considerable progress has now been made in unraveling the molecular structures of ATP and ADP hydrolyzing ecto-nucleotidases. The first enzyme identified was an ATP diphosphohydrolase (ecto-apyrase) that can hydrolyze either ATP or ADP. Human placental ATP diphosphohydrolase [5] is identical to the lymphoid cell activation antigen CD39 [6]. Expression of human CD39 in COS-7 cells leads to an increase in surface-located ATPase and ADPase activity [7,8]. Moreover, recombinant human and mouse CD39 inhibits ADP-induced platelet aggregation [8,9]. The prima...

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In the adult nervous system, multipotential stem cells of the subventricular zone of the lateral ventricles generate neuron precursors (type-A cells) that migrate via the rostral migratory stream to the olfactory bulb where they differentiate into neurons. The migrating neuroblasts are surrounded by a sheath of astrocytes (type-B cells). Using immunostaining, in situ hybridization and enzyme histochemistry, we demonstrate that the ecto-ATPase nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) is expressed in the subventricular zone and the rostral migratory stream of the adult rat brain. This enzyme hydrolyses extracellular nucleoside triphosphates to the respective nucleoside diphosphates and is thought to directly modulate ATP receptor-mediated cell communication. Double labelling for the astrocyte intermediate filament protein GFAP and the glial glutamate transporter GLAST identifies the NTPDase2-positive cells as type-B cells. During development the enzyme protein is first detected at E18, long before expression of the astrocyte marker GFAP. It gradually becomes expressed along the ventricular and subventricular zone of the brain, followed by complete retraction to the adult expression pattern at P21. NTPDase2 is transiently expressed in the outer molecular layer of the dentate gyrus and within the cerebellar white matter and is associated with select microvessels, tanycytes of the third ventricle, and subpial astrocytes of the adult brain. Our results suggest that NTPDase2 can serve as a novel marker for specifying subsets of cells during in vivo and in vitro studies of neural development and raise the possibility that ATP-mediated signalling pathways play a role in neural development and differentiation.

A short ischemic period induced by the transient occlusion of major brain arteries induces neuronal damage in selectively vulnerable regions of the hippocampus. Adenosine is considered to be one of the major neuroprotective substances produced in the ischemic brain. It can be released from damaged cells, but it also could be generated extracellularly from released ATP via a surface-located enzyme chain. Using the rat model of global forebrain ischemia, we applied a short (10 min) transient interruption of blood flow and studied the distribution of ectonucleotidase activities in the hippocampus. Northern hybridization of mRNA isolated from hippocampi of shamoperated and ischemic animals revealed an upregulation of ectoapyrase (capable of hydrolyzing nucleoside 5Ј-tri-and diphosphates) and ecto-5Ј-nucleotidase (capable of hydrolyzing nucleoside 5Ј-monophosphates). A histochemical analysis that used ATP, UTP, ADP, or AMP as substrates revealed a strong and selective increase in enzyme activity in the injured areas of the hippocampus. Enhanced staining could be observed first at 2 d. Staining increased within the next days and persisted at 28 d after ischemia. The spatiotemporal development of catalytic activities was identical for all substrates. It was most pronounced in the CA1 subfield and also could be detected in the dentate hilus and to a marginal extent in CA3. The histochemical staining corresponded closely to the development of markers for reactive glia, in particular of microglia. The upregulation of ectonucleotidase activities implies increased nucleotide release from the damaged tissue and could play a role in the postischemic control of nucleotide-mediated cellular responses.