J. Sévigny | Université Laval (original) (raw)

Papers by J. Sévigny

Research paper thumbnail of Generation and Characterization of Specific Antibodies to the Murine and Human Ectonucleotidase NTPDase8

Frontiers in pharmacology, 2017

The ectonucleotidase nucleoside triphosphate diphosphohydrolase-8 (NTPDase8) is the last member o... more The ectonucleotidase nucleoside triphosphate diphosphohydrolase-8 (NTPDase8) is the last member of the Ecto-NTPDase family to be discovered and characterized. It is a transmembrane protein which regulates the concentration of the agonists of P1 and P2 receptors at the cell surface. The functions of the enzyme are still not known partly due to the lack of specific tools such as antibodies. In this work, guinea pig polyclonal antibodies against mouse NTPDase8 and mouse monoclonal antibodies against human NTPDase8 have been generated and characterized. For the production of antibodies against mouse NTPDase8 several techniques have been tried. Several peptide antigens in several hosts (rabbit, rat, hamster, and guinea pig) failed to give a positive reaction suggesting that NTPDase8 is poorly immunogenic. In this study, we describe the successful process that led to anti-mouse NTPDase8, namely the cDNA immunization technique. Monoclonal antibodies to human NTPDase8 were also obtained by ...

Research paper thumbnail of Ectonucleotidases in the digestive system: focus on NTPDase3 localization

AJP: Gastrointestinal and Liver Physiology, 2011

Extracellular nucleotides and adenosine are biologically active molecules that bind members of th... more 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, immunohist...

Research paper thumbnail of Attenuated allergic airway inflammation in Cd39 null mice

Allergy, 2013

Background-Extracellular ATP is known to accumulate in the lung, following allergen challenge, an... more Background-Extracellular ATP is known to accumulate in the lung, following allergen challenge, and contributes via activation of purinergic receptors on dendritic cells (DC), to the development of allergic airway inflammation (AAI). Extracellular ATP-levels in the airways are normally tightly regulated by CD39. This ectonucleotidase is highly expressed by DC purified from skin (Langerhans cells) and bone marrow, and has been shown to modulate DC adaptive/ haptenic immune responses. In this study, we have evaluated the impact of Cd39 deletion and associated perturbation of purinergic signaling in AAI. Methods-Standard OVA-alum and house dust mite (HDM) bone marrow derived DC (BMDC) dependent models of AAI were used to study effects of Cd39. Migration assays, time lapse microscopy and T-cell priming assays were further used to determine functional relevance of Cd39 expression on BMDC in the setting of immune and Th2-mediated responses in these models. Results-Cd39 −/− mice exhibited marked increases in BALF ATP-levels but paradoxically exhibited limited AAI in both OVA-alum and HDM models. These pathophysiological abnormalities were associated with decreased myeloid DC activation and chemotaxis towards ATP, and were linked to purinergic receptor desensitization responses. Further, Cd39 −/− DCs exhibited limited capacity to both prime Th2-responses and form stable immune synaptic interactions with OVA-transgenic naïve T-cells.

Research paper thumbnail of Changes in expression and activity levels of ecto-5′-nucleotidase/CD73 along the mouse female estrous cycle

Acta Physiologica, 2010

Aim: Extracellular ATP and its hydrolysis product adenosine modulate various reproductive functio... more Aim: Extracellular ATP and its hydrolysis product adenosine modulate various reproductive functions such as those requiring contraction, hormone synthesis and maintenance of fluid composition. Moreover, adenosine is a key molecule for sperm capacitation. Extracellular nucleotide and nucleoside levels are affected by cell surface ectonucleotidases, amongst which the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family is the most abundant and effective to hydrolyse ATP and ADP to AMP. In the female reproductive tract three members of this family have been recently identified: NTPDase1, NTPDase2 and NTPDase3 (Histochem. Cell Biol. 131, 2009, 615). The purpose of the present study was to characterize in this system the expression profile of ecto-5¢-nucleotidase (CD73), the enzyme generating adenosine from AMP. Methods: Immunological techniques and in situ enzymatic assays were used to characterize the ecto-5¢-nucleotidase expression in the mouse female reproductive tract along the four stages of the estrous cycle, that were determined by vaginal smear examination. Results: Ecto-5¢-nucleotidase was abundantly detected in the corpora lutea of the ovaries, as well as in several epithelia, such as that of oviducts, uterus and endometrial glands. Marked changes in endometrial ecto-5¢-nucleotidase expression and activity along the estrous cycle are described, these being maximum at estrus phase, coinciding with optimal female sexual receptivity. Conclusion: The adenosine generated thereby, besides other functions, might contribute to sperm capacitation, thus significantly influencing fertility.

Research paper thumbnail of Ghrelin Is Produced In Taste Cells and Ghrelin Receptor Null Mice Show Reduced Taste Responsivity to Salty (NaCl) and Sour (Citric Acid) Tastants

PloS one, 2010

Background: The gustatory system plays a critical role in determining food preferences, food inta... more Background: The gustatory system plays a critical role in determining food preferences, food intake and energy balance. The exact mechanisms that fine tune taste sensitivity are currently poorly defined, but it is clear that numerous factors such as efferent input and specific signal transduction cascades are involved. Methodology/Principal Findings: Using immunohistochemical analyses, we show that ghrelin, a hormone classically considered to be an appetite-regulating hormone, is present within the taste buds of the tongue. Prepro-ghrelin, prohormone convertase 1/3 (PC 1/3), ghrelin, its cognate receptor (GHSR), and ghrelin-O-acyltransferase (GOAT , the enzyme that activates ghrelin) are expressed in Type I, II, III and IV taste cells of mouse taste buds. In addition, ghrelin and GHSR colocalize in the same taste cells, suggesting that ghrelin works in an autocrine manner in taste cells. To determine a role for ghrelin in modifying taste perception, we performed taste behavioral tests using GHSR null mice. GHSR null mice exhibited significantly reduced taste responsivity to sour (citric acid) and salty (sodium chloride) tastants. Conclusions/Significance: These findings suggest that ghrelin plays a local modulatory role in determining taste bud signaling and function and could be a novel mechanism for the modulation of salty and sour taste responsivity.

Research paper thumbnail of Identification and characterization of CD39/vascular ATP diphosphohydrolase

Journal of Biological …, 1996

Vascular ATP diphosphohydrolase (ATPDase) is a plasma membrane-bound enzyme that hydrolyses extra... more Vascular ATP diphosphohydrolase (ATPDase) is a plasma membrane-bound enzyme that hydrolyses extracellular ATP and ADP to AMP. Analysis of amino acid sequences available from various mammalian and avian ATPDases revealed their close homology with CD39, a putative B-cell activation marker. We, therefore, isolated CD39 cDNA from human endothelial cells and expressed this in COS-7 cells. CD39 was found to have both immunological identity to, and functional characteristics of, the vascular ATPDase. We also demonstrated that ATPDase could inhibit platelet aggregation in response to ADP, collagen, and thrombin, and that this activity in transfected COS-7 cells was lost following exposure to oxidative stress. ATPDase mRNA was present in human placenta, lung, skeletal muscle, kidney, and heart and was not detected in brain. Multiple RNA bands were detected with the CD39 cDNA probe that most probably represent different splicing products. Finally, we identified an unique conserved motif, DLG-GASTQ, that could be crucial for nucleotide binding, activity, and/or structure of ATPDase. Because ATPDase activity is lost with endothelial cell activation, overexpression of the functional enzyme, or a truncated mutant thereof, may prevent platelet activation associated with vascular inflammation.

Research paper thumbnail of Identification and Characterization of a Novel Hepatic

Research paper thumbnail of Purinergic Signalling in Immune System Regulation in Health and Disease

Mediators of Inflammation, 2015

The concept of a purinergic signalling system was first proposed by Professor Geoffrey Burnstock ... more The concept of a purinergic signalling system was first proposed by Professor Geoffrey Burnstock over 30 years ago. This includes the cellular responses to purine nucleotides, such as ATP, and nucleosides, such as adenosine, that act as extracellular messengers playing a role through specific nucleotide and adenosine receptors in all systems. Indeed, in addition to their role in cellular metabolism, nucleotides as well as nucleosides are extracellular mediators that activate biological responses in all cells. Cells subjected to activation or shear or mechanical stress release nucleotides such as ATP, ADP, UTP, and UDP in large amounts. All cells can release nucleotides in a controlled fashion . The mechanisms of nucleotide release have been the focus of intense research activities but are still not fully understood. While activated platelets and neurons release nucleotides by exocytosis, neutrophils, and T lymphocytes use pannexin-1 hemichannels for nucleotide efflux, some cells also constitutively release nucleotides.

Research paper thumbnail of Ecto-5'-Nucleotidase (CD73)-Mediated Formation of Adenosine Is Critical for the Striatal Adenosine A2A Receptor Functions

Journal of Neuroscience, 2013

Adenosine is a neuromodulator acting through inhibitory A 1 receptors (A 1 Rs) and facilitatory A... more Adenosine is a neuromodulator acting through inhibitory A 1 receptors (A 1 Rs) and facilitatory A 2A Rs, which have similar affinities for adenosine. It has been shown that the activity of intracellular adenosine kinase preferentially controls the activation of A 1 Rs, but the source of the adenosine activating A 2A Rs is unknown. We now show that ecto-5Ј-nucleotidase (CD73), the major enzyme able to convert extracellular AMP into adenosine, colocalizes with A 2A Rs in the basal ganglia. In addition to astrocytes, striatal CD73 is prominently localized to postsynaptic sites. Notably, CD73 coimmunoprecipitated with A 2A Rs and proximity ligation assays confirmed the close proximity of CD73 and A 2A Rs in the striatum. Accordingly, the cAMP formation in synaptosomes as well as the hypolocomotion induced by a novel A 2A R prodrug that requires CD73 metabolization to activate A 2A Rs were observed in wild-type mice, but not in CD73 knock-out (KO) mice or A 2A R KO mice. Moreover, CD73 KO mice displayed increased working memory performance and a blunted amphetamineinduced sensitization, mimicking the phenotype of global or forebrain-A 2A R KO mice, as well as upon pharmacological A 2A R blockade. These results show that CD73-mediated formation of extracellular adenosine is responsible for the activation of striatal A 2A R function. This study points to CD73 as a new target that can fine-tune A 2A R activity, and a novel therapeutic target to manipulate A 2A R-mediated control of striatal function and neurodegeneration.

Research paper thumbnail of Distribution of ecto-nucleotidases in mouse sensory circuits suggests roles for nucleoside triphosphate diphosphohydrolase-3 in nociception and mechanoreception

Neuroscience, 2011

Nucleotide-activated P2X channels and P2Y metabotropic receptors participate in nociceptive signa... more Nucleotide-activated P2X channels and P2Y metabotropic receptors participate in nociceptive signaling. Agonist availability is regulated by nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), -2, -3, and -8, a family of enzymes that hydrolyze extracellular ATP to generate ADP (a P2Y agonist) and AMP. They provide a major source of extracellular AMP, the substrate for adenosine production by ecto-5=-nucleotidase (NT5E), and thereby regulate adenosine (P1) receptor signaling. NTPDases vary in their efficiency of tri-and diphosphate hydrolysis; therefore, which family members are expressed impacts nucleotide availability and half-life. This study employed enzyme activity histochemistry to examine the distribution of ATPase activity and immunohistochemistry for NTPDase1, 2, 3, and 8 in dorsal root ganglion (DRG) and spinal cord. Nucleotidase activity was robust in spinal dorsal horn, confirming that nociceptive pathways are a major site of nucleotide transmission. In DRG, extensive staining revealed ATPase activity in a subset of neurons and in non-neuronal cells. mRNA for NTPDase1-3, but not NTPDase8, was detected in lumbar DRG and spinal cord. Immunoreactivity for NTPDase3 closely matched the distribution of ATPase activity, labeling DRG central projections in the dorsal root and superficial dorsal horn, as well as intrinsic spinal neurons concentrated in lamina II. In DRG, NTPDase3 co-localized with markers of nociceptors and with NT5E. In addition, labeling of a subset of larger-diameter neurons in DRG was consistent with intense staining of Meissner corpuscle afferents in glabrous skin. Merkel cells and terminal Schwann cells of hair follicle afferents were also labeled, but the axons themselves were negative. We propose that NTPDase3 is a key regulator of nociceptive signaling that also makes an unexpected contribution to innocuous tactile sensation.

Research paper thumbnail of Role of ecto-NTPDases on UDP-sensitive P2Y 6 receptor activation during osteogenic differentiation of primary bone marrow stromal cells from postmenopausal women

Journal of Cellular Physiology, 2012

This study aimed at investigating the expression and function of uracil nucleotide-sensitive rece... more This study aimed at investigating the expression and function of uracil nucleotide-sensitive receptors (P2Y 2 , P2Y 4 , and P2Y 6 ) on osteogenic differentiation of human bone marrow stromal cells (BMSCs) in culture. Bone marrow specimens were obtained from postmenopausal female patients (68 AE 5 years old, n ¼ 18) undergoing total hip arthroplasty. UTP and UDP (100 mM) facilitated osteogenic differentiation of the cells measured as increases in alkaline phosphatase (ALP) activity, without affecting cell proliferation. Uracil nucleotides concentration-dependently increased [Ca 2þ ] i in BMSCs; their effects became less evident with time (7 > 21 days) of the cells in culture. Selective activation of P2Y 6 receptors with the stable UDP analog, PSB 0474, mimicked the effects of both UTP and UDP, whereas UTPgS was devoid of effect. Selective blockade of P2Y 6 receptors with MRS 2578 prevented [Ca 2þ ] i rises and osteogenic differentiation caused by UDP at all culture time points. BMSCs are immunoreactive against P2Y 2 , P2Y 4 , and P2Y 6 receptors. While the expression of P2Y 6 receptors remained fairly constant (7$21 days), P2Y 2 and P2Y 4 became evident only in less proliferative and more differentiated cultures (7 < 21 days). The rate of extracellular UTP and UDP inactivation was higher in less proliferative and more differentiated cell populations. Immunoreactivity against NTPDase1, -2, and -3 rises as cells differentiate (7 < 21 days). Data show that uracil nucleotides are important regulators of osteogenic cells differentiation predominantly through the activation of UDP-sensitive P2Y 6 receptors coupled to increases in [Ca 2þ ] i . Endogenous actions of uracil nucleotides may be balanced through specific NTPDases determining whether osteoblast progenitors are driven into proliferation or differentiation.

Research paper thumbnail of Transforming growth factor-β and substrate stiffness regulate portal fibroblast activation in culture

Hepatology, 2007

Myofibroblasts derived from portal fibroblasts are important fibrogenic cells in the early stages... more Myofibroblasts derived from portal fibroblasts are important fibrogenic cells in the early stages of biliary fibrosis. In contrast to hepatic stellate cells, portal fibroblasts have not been well studied in vitro, and little is known about their myofibroblastic differentiation. In this article we report the isolation and characterization of rat portal fibroblasts in culture. We demonstrate that primary portal fibroblasts undergo differentiation to ␣-smooth muscle actin-expressing myofibroblasts over 10 -14 days. Marker analysis comparing portal fibroblasts to hepatic stellate cells demonstrated that these are distinct populations and that staining with elastin and desmin can differentiate between them. Portal fibroblasts expressed elastin at all stages in culture but never expressed desmin, whereas hepatic stellate cells consistently expressed desmin but never elastin. Immunostaining of rat liver tissue confirmed these results in vivo.

Research paper thumbnail of Ca2+ Responses in Enteric Glia Are Mediated by Connexin-43 Hemichannels and Modulate Colonic Transit in Mice

Gastroenterology, 2014

In the enteric nervous system, neurotransmitters initiate changes in calcium (Ca 2þ responses) in... more In the enteric nervous system, neurotransmitters initiate changes in calcium (Ca 2þ responses) in glia, but it is not clear how this process affects intestinal function. We investigated whether Ca 2þ -mediated responses in enteric glia are required to maintain gastrointestinal function. METHODS: We used in situ Ca 2þ imaging to monitor glial Ca 2þ responses, which were manipulated with pharmacologic agents or via gliaspecific disruption of the gene encoding connexin-43 (Cx43) (hGFAP::CreER T2þ/À /Cx43 f/f mice). Gastrointestinal function was assessed based on pellet output, total gut transit, colonic bead expulsion, and muscle tension recordings. Proteins were localized and quantified by immunohistochemistry, immunoblot, and reverse transcription polymerase chain reaction analyses. RESULTS: Ca 2þ responses in enteric glia of mice were mediated by Cx43 hemichannels. Cx43 immunoreactivity was confined to enteric glia within the myenteric plexus of the mouse colon; the Cx43 inhibitors carbenoxolone and 43Gap26 inhibited the ability of enteric glia to propagate Ca 2þ responses. In vivo attenuation of Ca 2þ responses in the enteric glial network slowed gut transit overall and delayed colonic transit-these changes are also observed during normal aging. Altered motility with increasing age was associated with reduced glial Ca 2þmediated responses and changes in glial expression of Cx43 messenger RNA and protein. CONCLUSIONS: Ca 2þ -mediated responses in enteric glia regulate gastrointestinal function in mice. Altered intercellular signaling between enteric glia and neurons might contribute to motility disorders.

Research paper thumbnail of Synaptic and Subsynaptic Localization of NTPDASES 1,2,3,and ECTO- 5’NUCLEOTIDASE in the Rat Hippocampus and Striatum

Frontiers in Neuroscience, 1970

Research paper thumbnail of Purification of pancreas type-I ATP diphosphohydrolase and identification by affinity labelling with the 5'-p-fluorosulphonylbenzoyladenosine ATP analogue

The Biochemical journal, 1995

The enzyme recently identified as type-I ATP diphosphohydrolase (ATPDase; EC 3.6.1.5) has been pu... more The enzyme recently identified as type-I ATP diphosphohydrolase (ATPDase; EC 3.6.1.5) has been purified from the zymogen granule membrane of pig pancreas. After solubilization with Triton X-100 and chromatographies on ion-exchange and Affi-Gel Blue columns an approximate 3500-fold purification was obtained. The enzyme preparation with a specific activity of 45 units/mg of protein was much further purified by PAGE under non-denaturing conditions. The active band localized on the gel contained two proteins after SDS/PAGE and silver staining, corresponding to apparent molecular masses of 56 and 54 kDa. The identity of the ATPDase was confirmed by an affinity labelling technique with 5'-p-fluorosulphonylbenzoyladenosine (FSBA) as an ATP analogue. The latter was detected by a Western blot technique. A strong reaction was observed with the band corresponding to 54 kDa. N-terminal sequence analysis revealed that the 56 kDa protein has significant similarities (50-72%) with lipases, whe...

Research paper thumbnail of Nucleoside triphosphate diphosphohydrolases role in the pathophysiology of cognitive impairment induced by seizure in early age

Neuroscience, 2011

Background: Ecto-Nucleoside Triphosphate Diphosphohydrolases (Ecto-NTPDases) are enzymes that hyd... more Background: Ecto-Nucleoside Triphosphate Diphosphohydrolases (Ecto-NTPDases) are enzymes that hydrolyze tri-and/or di-phosphate nucleotides. Evidences point to their participation in Trypanosoma cruzi virulence and infectivity. In this work, we evaluate TcNTPDase-1 gene expression in comparison with ecto-NTPDase activity, in order to study the role of TcNTPDase-1 in parasite virulence, infectivity and adaptation to heat shock. Findings: Comparison between distinct T. cruzi isolates (Y, 3663 and 4167 strains, and Dm28c, LL014 and CL-14 clones) showed that TcNTPDase-1 expression was 7.2 ± 1.5 times higher in the Dm28c than the CL-14 avirulent clone. A remarkable expression increase was also observed in the trypomastigote and amastigote forms (22.5 ± 5.6 and 16.3 ± 3.8 times higher than epimastigotes, respectively), indicating that TcNTPDase-1 is overexpressed in T. cruzi infective forms. Moreover, heat shock and long-term cultivation also induced a significant increment on TcNTPDase-1 expression.

Research paper thumbnail of Diadenosine 5′,5′′-(Boranated)polyphosphonate Analogues as Selective Nucleotide Pyrophosphatase/Phosphodiesterase Inhibitors ⊥

Journal of Medicinal Chemistry, 2010

Nucleotide pyrophosphatase/phosphodiesterases (NPPs) hydrolyze extracellular nucleotides and dinu... more Nucleotide pyrophosphatase/phosphodiesterases (NPPs) hydrolyze extracellular nucleotides and dinucleotides and thus control purinergic signaling. Enhanced NPP activity is implicated in health disorders such as osteoarthritis and cancer. We designed novel diadenosine polyphosphonate derivatives as potential NPP inhibitors. Analogues 1-4 bear a phosphonate and/or boranophosphate group and/or a 2&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-H atom instead of a 2&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-OH group. In comparison to ATP, analogues 1-4 were barely hydrolyzed by human NTPDase1, -2, -3, and -8 (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;5% hydrolysis) and NPP1 and -3 (≤ 13%) and were not hydrolyzed by ecto-5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-nucleotidase, unlike AMP. These derivatives did not affect NTPDase activity, and analogues 1 and 2 did not inhibit ecto-5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-nucleotidase. All analogues blocked ∼80% of the NPP2-dependent hydrolysis of pnp-TMP, a specific NPP substrate, and inhibited the catabolism of pnp-TMP (K(i) and IC₅₀ both found to be between 10 and 60 μM), Ap₅A, and ATP by NPP1. The activity of NPP3 was inhibited to a lesser extent by the new analogues, with compounds 1 and 4 being the most effective in that respect. The analogues dramatically reduced the level of hydrolysis of pnp-TMP at the cell surface of both osteocarcinoma and colon cancer cells. Importantly, analogues 1-4 exhibited significantly reduced agonistic activity toward human P2Y₁,₁₁) receptors (except for analogue 1) and no activity with human P2Y₂ receptor. Our data provide strong evidence that analogue 2 is the first specific NPP inhibitor to be described.

Research paper thumbnail of Diadenosine and Diuridine Poly(borano)phosphate Analogues:  Synthesis, Chemical and Enzymatic Stability, and Activity at P2Y 1 and P2Y 2 Receptors †

Journal of Medicinal Chemistry, 2006

Dinucleoside polyphosphates, Np n N′, exert their physiological effects via P2 receptors. They ar... more Dinucleoside polyphosphates, Np n N′, exert their physiological effects via P2 receptors. They are attractive drug targets as they offer better stability and specificity compared to nucleotides, the most common P2receptor ligands. To further improve the properties of Np n N′, which are still pharmacologically unsatisfactory, we developed novel boranophosphate isosteres of dinucleoside polyphosphates, denoted as Np n (B)N. These analogues were obtained in a facile and efficient synthesis as the exclusive products in a concerted reaction of two nucleoside phosphorimidazolides and inorganic boranophosphate. This unusual reaction is due to the preorganization of three reactant molecules by the Mg 2+ ion. We found that Ap 3/5 ( /γ-B)A analogues were potent P2Y 1 -R agonists. Ap 5 (γ-B)A was equipotent to 2-MeS-ADP (EC 50 6.3 × 10 -8 M), thus making it one of the most potent P2Y 1 -R agonists currently known. Moreover, Ap 5 (γ-B)A did not activate P2Y 2 -R. In contrast, Up 3/5 ( /γ-B)U analogues were extremely poor agonists of both P2Y 1 -R and P2Y 2 -R. Np n (B)N analogues exhibited remarkable chemical stability under physiological conditions. Under conditions mimicking gastric juice, Np 3 ( -B)N analogues exhibited a half-life (t 1/2 ) of 1.3 h, whereas Np 5 (γ-B)N degraded at a much faster rate (t 1/2 18 min). The hydrolysis of Ap 3 ( -B)A by human nucleotide pyrophosphatase phosphodiesterases (NPP1 and NPP3) was slowed by 40% and 59%, respectively, as compared to Ap 3 A. However, this effect of the boranophosphate was position-dependent, as Np 5 (γ-B)N was degraded at a rate comparable to that of Np 5 N. In summary, Ap 5 (γ-B)A appears to be a highly potent and selective P2Y 1 -R agonist, as compared to the parent compound. This promising scaffold will be applied in the design of future metabolically stable analogues. † A U.S. patent was filed June 15, 2005.

Research paper thumbnail of Extracellular nucleotides mediate LPS-induced neutrophil migration in vitro and in vivo

Journal of Leukocyte Biology, 2007

Extracellular nucleotides are emerging as important inflammatory mediators. Here, we demonstrate ... more Extracellular nucleotides are emerging as important inflammatory mediators. Here, we demonstrate that these molecules mediate LPSinduced neutrophil migration in vitro and in vivo. Apyrase, a nucleotide scavenger, reduced the ability of LPS-stimulated monocytes to recruit neutrophils, as assayed using a modified Boyden chamber. This effect resulted from the inhibition of IL-8 release from monocytes. Furthermore, LPS-induced IL-8 release by monocytes was attenuated significantly by P2Y 6 receptor antagonists, RB-2 and MRS2578. Reciprocally, UDP, the selective P2Y 6 agonist, induced IL-8 release by monocytes. As for LPS, the media of UDP-stimulated monocytes were chemotactic for neutrophils; IL-8 accounted for ϳ50% of neutrophil migration induced by the media of LPS-or UDP-treated monocytes in transendothelial migration assays. It is important that in the murine air-pouch model, extracellular nucleotides were instrumental in LPSinduced neutrophil migration. Altogether, these data imply that LPS induces the release of nucleotides from monocytes and that by autocrine stimulation, the latter molecules regulate neutrophil migration caused by Gram-negative bacteria, suggesting a proinflammatory role of extracellular nucleotides in innate immunity. J. Leukoc. Biol. 81: 1269 -1275; 2007.

Research paper thumbnail of Nucleoside triphosphate diphosphohydrolase-2 is the ecto-ATPase of type I cells in taste buds

The Journal of Comparative Neurology, 2006

Research paper thumbnail of Generation and Characterization of Specific Antibodies to the Murine and Human Ectonucleotidase NTPDase8

Frontiers in pharmacology, 2017

The ectonucleotidase nucleoside triphosphate diphosphohydrolase-8 (NTPDase8) is the last member o... more The ectonucleotidase nucleoside triphosphate diphosphohydrolase-8 (NTPDase8) is the last member of the Ecto-NTPDase family to be discovered and characterized. It is a transmembrane protein which regulates the concentration of the agonists of P1 and P2 receptors at the cell surface. The functions of the enzyme are still not known partly due to the lack of specific tools such as antibodies. In this work, guinea pig polyclonal antibodies against mouse NTPDase8 and mouse monoclonal antibodies against human NTPDase8 have been generated and characterized. For the production of antibodies against mouse NTPDase8 several techniques have been tried. Several peptide antigens in several hosts (rabbit, rat, hamster, and guinea pig) failed to give a positive reaction suggesting that NTPDase8 is poorly immunogenic. In this study, we describe the successful process that led to anti-mouse NTPDase8, namely the cDNA immunization technique. Monoclonal antibodies to human NTPDase8 were also obtained by ...

Research paper thumbnail of Ectonucleotidases in the digestive system: focus on NTPDase3 localization

AJP: Gastrointestinal and Liver Physiology, 2011

Extracellular nucleotides and adenosine are biologically active molecules that bind members of th... more 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, immunohist...

Research paper thumbnail of Attenuated allergic airway inflammation in Cd39 null mice

Allergy, 2013

Background-Extracellular ATP is known to accumulate in the lung, following allergen challenge, an... more Background-Extracellular ATP is known to accumulate in the lung, following allergen challenge, and contributes via activation of purinergic receptors on dendritic cells (DC), to the development of allergic airway inflammation (AAI). Extracellular ATP-levels in the airways are normally tightly regulated by CD39. This ectonucleotidase is highly expressed by DC purified from skin (Langerhans cells) and bone marrow, and has been shown to modulate DC adaptive/ haptenic immune responses. In this study, we have evaluated the impact of Cd39 deletion and associated perturbation of purinergic signaling in AAI. Methods-Standard OVA-alum and house dust mite (HDM) bone marrow derived DC (BMDC) dependent models of AAI were used to study effects of Cd39. Migration assays, time lapse microscopy and T-cell priming assays were further used to determine functional relevance of Cd39 expression on BMDC in the setting of immune and Th2-mediated responses in these models. Results-Cd39 −/− mice exhibited marked increases in BALF ATP-levels but paradoxically exhibited limited AAI in both OVA-alum and HDM models. These pathophysiological abnormalities were associated with decreased myeloid DC activation and chemotaxis towards ATP, and were linked to purinergic receptor desensitization responses. Further, Cd39 −/− DCs exhibited limited capacity to both prime Th2-responses and form stable immune synaptic interactions with OVA-transgenic naïve T-cells.

Research paper thumbnail of Changes in expression and activity levels of ecto-5′-nucleotidase/CD73 along the mouse female estrous cycle

Acta Physiologica, 2010

Aim: Extracellular ATP and its hydrolysis product adenosine modulate various reproductive functio... more Aim: Extracellular ATP and its hydrolysis product adenosine modulate various reproductive functions such as those requiring contraction, hormone synthesis and maintenance of fluid composition. Moreover, adenosine is a key molecule for sperm capacitation. Extracellular nucleotide and nucleoside levels are affected by cell surface ectonucleotidases, amongst which the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family is the most abundant and effective to hydrolyse ATP and ADP to AMP. In the female reproductive tract three members of this family have been recently identified: NTPDase1, NTPDase2 and NTPDase3 (Histochem. Cell Biol. 131, 2009, 615). The purpose of the present study was to characterize in this system the expression profile of ecto-5¢-nucleotidase (CD73), the enzyme generating adenosine from AMP. Methods: Immunological techniques and in situ enzymatic assays were used to characterize the ecto-5¢-nucleotidase expression in the mouse female reproductive tract along the four stages of the estrous cycle, that were determined by vaginal smear examination. Results: Ecto-5¢-nucleotidase was abundantly detected in the corpora lutea of the ovaries, as well as in several epithelia, such as that of oviducts, uterus and endometrial glands. Marked changes in endometrial ecto-5¢-nucleotidase expression and activity along the estrous cycle are described, these being maximum at estrus phase, coinciding with optimal female sexual receptivity. Conclusion: The adenosine generated thereby, besides other functions, might contribute to sperm capacitation, thus significantly influencing fertility.

Research paper thumbnail of Ghrelin Is Produced In Taste Cells and Ghrelin Receptor Null Mice Show Reduced Taste Responsivity to Salty (NaCl) and Sour (Citric Acid) Tastants

PloS one, 2010

Background: The gustatory system plays a critical role in determining food preferences, food inta... more Background: The gustatory system plays a critical role in determining food preferences, food intake and energy balance. The exact mechanisms that fine tune taste sensitivity are currently poorly defined, but it is clear that numerous factors such as efferent input and specific signal transduction cascades are involved. Methodology/Principal Findings: Using immunohistochemical analyses, we show that ghrelin, a hormone classically considered to be an appetite-regulating hormone, is present within the taste buds of the tongue. Prepro-ghrelin, prohormone convertase 1/3 (PC 1/3), ghrelin, its cognate receptor (GHSR), and ghrelin-O-acyltransferase (GOAT , the enzyme that activates ghrelin) are expressed in Type I, II, III and IV taste cells of mouse taste buds. In addition, ghrelin and GHSR colocalize in the same taste cells, suggesting that ghrelin works in an autocrine manner in taste cells. To determine a role for ghrelin in modifying taste perception, we performed taste behavioral tests using GHSR null mice. GHSR null mice exhibited significantly reduced taste responsivity to sour (citric acid) and salty (sodium chloride) tastants. Conclusions/Significance: These findings suggest that ghrelin plays a local modulatory role in determining taste bud signaling and function and could be a novel mechanism for the modulation of salty and sour taste responsivity.

Research paper thumbnail of Identification and characterization of CD39/vascular ATP diphosphohydrolase

Journal of Biological …, 1996

Vascular ATP diphosphohydrolase (ATPDase) is a plasma membrane-bound enzyme that hydrolyses extra... more Vascular ATP diphosphohydrolase (ATPDase) is a plasma membrane-bound enzyme that hydrolyses extracellular ATP and ADP to AMP. Analysis of amino acid sequences available from various mammalian and avian ATPDases revealed their close homology with CD39, a putative B-cell activation marker. We, therefore, isolated CD39 cDNA from human endothelial cells and expressed this in COS-7 cells. CD39 was found to have both immunological identity to, and functional characteristics of, the vascular ATPDase. We also demonstrated that ATPDase could inhibit platelet aggregation in response to ADP, collagen, and thrombin, and that this activity in transfected COS-7 cells was lost following exposure to oxidative stress. ATPDase mRNA was present in human placenta, lung, skeletal muscle, kidney, and heart and was not detected in brain. Multiple RNA bands were detected with the CD39 cDNA probe that most probably represent different splicing products. Finally, we identified an unique conserved motif, DLG-GASTQ, that could be crucial for nucleotide binding, activity, and/or structure of ATPDase. Because ATPDase activity is lost with endothelial cell activation, overexpression of the functional enzyme, or a truncated mutant thereof, may prevent platelet activation associated with vascular inflammation.

Research paper thumbnail of Identification and Characterization of a Novel Hepatic

Research paper thumbnail of Purinergic Signalling in Immune System Regulation in Health and Disease

Mediators of Inflammation, 2015

The concept of a purinergic signalling system was first proposed by Professor Geoffrey Burnstock ... more The concept of a purinergic signalling system was first proposed by Professor Geoffrey Burnstock over 30 years ago. This includes the cellular responses to purine nucleotides, such as ATP, and nucleosides, such as adenosine, that act as extracellular messengers playing a role through specific nucleotide and adenosine receptors in all systems. Indeed, in addition to their role in cellular metabolism, nucleotides as well as nucleosides are extracellular mediators that activate biological responses in all cells. Cells subjected to activation or shear or mechanical stress release nucleotides such as ATP, ADP, UTP, and UDP in large amounts. All cells can release nucleotides in a controlled fashion . The mechanisms of nucleotide release have been the focus of intense research activities but are still not fully understood. While activated platelets and neurons release nucleotides by exocytosis, neutrophils, and T lymphocytes use pannexin-1 hemichannels for nucleotide efflux, some cells also constitutively release nucleotides.

Research paper thumbnail of Ecto-5'-Nucleotidase (CD73)-Mediated Formation of Adenosine Is Critical for the Striatal Adenosine A2A Receptor Functions

Journal of Neuroscience, 2013

Adenosine is a neuromodulator acting through inhibitory A 1 receptors (A 1 Rs) and facilitatory A... more Adenosine is a neuromodulator acting through inhibitory A 1 receptors (A 1 Rs) and facilitatory A 2A Rs, which have similar affinities for adenosine. It has been shown that the activity of intracellular adenosine kinase preferentially controls the activation of A 1 Rs, but the source of the adenosine activating A 2A Rs is unknown. We now show that ecto-5Ј-nucleotidase (CD73), the major enzyme able to convert extracellular AMP into adenosine, colocalizes with A 2A Rs in the basal ganglia. In addition to astrocytes, striatal CD73 is prominently localized to postsynaptic sites. Notably, CD73 coimmunoprecipitated with A 2A Rs and proximity ligation assays confirmed the close proximity of CD73 and A 2A Rs in the striatum. Accordingly, the cAMP formation in synaptosomes as well as the hypolocomotion induced by a novel A 2A R prodrug that requires CD73 metabolization to activate A 2A Rs were observed in wild-type mice, but not in CD73 knock-out (KO) mice or A 2A R KO mice. Moreover, CD73 KO mice displayed increased working memory performance and a blunted amphetamineinduced sensitization, mimicking the phenotype of global or forebrain-A 2A R KO mice, as well as upon pharmacological A 2A R blockade. These results show that CD73-mediated formation of extracellular adenosine is responsible for the activation of striatal A 2A R function. This study points to CD73 as a new target that can fine-tune A 2A R activity, and a novel therapeutic target to manipulate A 2A R-mediated control of striatal function and neurodegeneration.

Research paper thumbnail of Distribution of ecto-nucleotidases in mouse sensory circuits suggests roles for nucleoside triphosphate diphosphohydrolase-3 in nociception and mechanoreception

Neuroscience, 2011

Nucleotide-activated P2X channels and P2Y metabotropic receptors participate in nociceptive signa... more Nucleotide-activated P2X channels and P2Y metabotropic receptors participate in nociceptive signaling. Agonist availability is regulated by nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), -2, -3, and -8, a family of enzymes that hydrolyze extracellular ATP to generate ADP (a P2Y agonist) and AMP. They provide a major source of extracellular AMP, the substrate for adenosine production by ecto-5=-nucleotidase (NT5E), and thereby regulate adenosine (P1) receptor signaling. NTPDases vary in their efficiency of tri-and diphosphate hydrolysis; therefore, which family members are expressed impacts nucleotide availability and half-life. This study employed enzyme activity histochemistry to examine the distribution of ATPase activity and immunohistochemistry for NTPDase1, 2, 3, and 8 in dorsal root ganglion (DRG) and spinal cord. Nucleotidase activity was robust in spinal dorsal horn, confirming that nociceptive pathways are a major site of nucleotide transmission. In DRG, extensive staining revealed ATPase activity in a subset of neurons and in non-neuronal cells. mRNA for NTPDase1-3, but not NTPDase8, was detected in lumbar DRG and spinal cord. Immunoreactivity for NTPDase3 closely matched the distribution of ATPase activity, labeling DRG central projections in the dorsal root and superficial dorsal horn, as well as intrinsic spinal neurons concentrated in lamina II. In DRG, NTPDase3 co-localized with markers of nociceptors and with NT5E. In addition, labeling of a subset of larger-diameter neurons in DRG was consistent with intense staining of Meissner corpuscle afferents in glabrous skin. Merkel cells and terminal Schwann cells of hair follicle afferents were also labeled, but the axons themselves were negative. We propose that NTPDase3 is a key regulator of nociceptive signaling that also makes an unexpected contribution to innocuous tactile sensation.

Research paper thumbnail of Role of ecto-NTPDases on UDP-sensitive P2Y 6 receptor activation during osteogenic differentiation of primary bone marrow stromal cells from postmenopausal women

Journal of Cellular Physiology, 2012

This study aimed at investigating the expression and function of uracil nucleotide-sensitive rece... more This study aimed at investigating the expression and function of uracil nucleotide-sensitive receptors (P2Y 2 , P2Y 4 , and P2Y 6 ) on osteogenic differentiation of human bone marrow stromal cells (BMSCs) in culture. Bone marrow specimens were obtained from postmenopausal female patients (68 AE 5 years old, n ¼ 18) undergoing total hip arthroplasty. UTP and UDP (100 mM) facilitated osteogenic differentiation of the cells measured as increases in alkaline phosphatase (ALP) activity, without affecting cell proliferation. Uracil nucleotides concentration-dependently increased [Ca 2þ ] i in BMSCs; their effects became less evident with time (7 > 21 days) of the cells in culture. Selective activation of P2Y 6 receptors with the stable UDP analog, PSB 0474, mimicked the effects of both UTP and UDP, whereas UTPgS was devoid of effect. Selective blockade of P2Y 6 receptors with MRS 2578 prevented [Ca 2þ ] i rises and osteogenic differentiation caused by UDP at all culture time points. BMSCs are immunoreactive against P2Y 2 , P2Y 4 , and P2Y 6 receptors. While the expression of P2Y 6 receptors remained fairly constant (7$21 days), P2Y 2 and P2Y 4 became evident only in less proliferative and more differentiated cultures (7 < 21 days). The rate of extracellular UTP and UDP inactivation was higher in less proliferative and more differentiated cell populations. Immunoreactivity against NTPDase1, -2, and -3 rises as cells differentiate (7 < 21 days). Data show that uracil nucleotides are important regulators of osteogenic cells differentiation predominantly through the activation of UDP-sensitive P2Y 6 receptors coupled to increases in [Ca 2þ ] i . Endogenous actions of uracil nucleotides may be balanced through specific NTPDases determining whether osteoblast progenitors are driven into proliferation or differentiation.

Research paper thumbnail of Transforming growth factor-β and substrate stiffness regulate portal fibroblast activation in culture

Hepatology, 2007

Myofibroblasts derived from portal fibroblasts are important fibrogenic cells in the early stages... more Myofibroblasts derived from portal fibroblasts are important fibrogenic cells in the early stages of biliary fibrosis. In contrast to hepatic stellate cells, portal fibroblasts have not been well studied in vitro, and little is known about their myofibroblastic differentiation. In this article we report the isolation and characterization of rat portal fibroblasts in culture. We demonstrate that primary portal fibroblasts undergo differentiation to ␣-smooth muscle actin-expressing myofibroblasts over 10 -14 days. Marker analysis comparing portal fibroblasts to hepatic stellate cells demonstrated that these are distinct populations and that staining with elastin and desmin can differentiate between them. Portal fibroblasts expressed elastin at all stages in culture but never expressed desmin, whereas hepatic stellate cells consistently expressed desmin but never elastin. Immunostaining of rat liver tissue confirmed these results in vivo.

Research paper thumbnail of Ca2+ Responses in Enteric Glia Are Mediated by Connexin-43 Hemichannels and Modulate Colonic Transit in Mice

Gastroenterology, 2014

In the enteric nervous system, neurotransmitters initiate changes in calcium (Ca 2þ responses) in... more In the enteric nervous system, neurotransmitters initiate changes in calcium (Ca 2þ responses) in glia, but it is not clear how this process affects intestinal function. We investigated whether Ca 2þ -mediated responses in enteric glia are required to maintain gastrointestinal function. METHODS: We used in situ Ca 2þ imaging to monitor glial Ca 2þ responses, which were manipulated with pharmacologic agents or via gliaspecific disruption of the gene encoding connexin-43 (Cx43) (hGFAP::CreER T2þ/À /Cx43 f/f mice). Gastrointestinal function was assessed based on pellet output, total gut transit, colonic bead expulsion, and muscle tension recordings. Proteins were localized and quantified by immunohistochemistry, immunoblot, and reverse transcription polymerase chain reaction analyses. RESULTS: Ca 2þ responses in enteric glia of mice were mediated by Cx43 hemichannels. Cx43 immunoreactivity was confined to enteric glia within the myenteric plexus of the mouse colon; the Cx43 inhibitors carbenoxolone and 43Gap26 inhibited the ability of enteric glia to propagate Ca 2þ responses. In vivo attenuation of Ca 2þ responses in the enteric glial network slowed gut transit overall and delayed colonic transit-these changes are also observed during normal aging. Altered motility with increasing age was associated with reduced glial Ca 2þmediated responses and changes in glial expression of Cx43 messenger RNA and protein. CONCLUSIONS: Ca 2þ -mediated responses in enteric glia regulate gastrointestinal function in mice. Altered intercellular signaling between enteric glia and neurons might contribute to motility disorders.

Research paper thumbnail of Synaptic and Subsynaptic Localization of NTPDASES 1,2,3,and ECTO- 5’NUCLEOTIDASE in the Rat Hippocampus and Striatum

Frontiers in Neuroscience, 1970

Research paper thumbnail of Purification of pancreas type-I ATP diphosphohydrolase and identification by affinity labelling with the 5'-p-fluorosulphonylbenzoyladenosine ATP analogue

The Biochemical journal, 1995

The enzyme recently identified as type-I ATP diphosphohydrolase (ATPDase; EC 3.6.1.5) has been pu... more The enzyme recently identified as type-I ATP diphosphohydrolase (ATPDase; EC 3.6.1.5) has been purified from the zymogen granule membrane of pig pancreas. After solubilization with Triton X-100 and chromatographies on ion-exchange and Affi-Gel Blue columns an approximate 3500-fold purification was obtained. The enzyme preparation with a specific activity of 45 units/mg of protein was much further purified by PAGE under non-denaturing conditions. The active band localized on the gel contained two proteins after SDS/PAGE and silver staining, corresponding to apparent molecular masses of 56 and 54 kDa. The identity of the ATPDase was confirmed by an affinity labelling technique with 5'-p-fluorosulphonylbenzoyladenosine (FSBA) as an ATP analogue. The latter was detected by a Western blot technique. A strong reaction was observed with the band corresponding to 54 kDa. N-terminal sequence analysis revealed that the 56 kDa protein has significant similarities (50-72%) with lipases, whe...

Research paper thumbnail of Nucleoside triphosphate diphosphohydrolases role in the pathophysiology of cognitive impairment induced by seizure in early age

Neuroscience, 2011

Background: Ecto-Nucleoside Triphosphate Diphosphohydrolases (Ecto-NTPDases) are enzymes that hyd... more Background: Ecto-Nucleoside Triphosphate Diphosphohydrolases (Ecto-NTPDases) are enzymes that hydrolyze tri-and/or di-phosphate nucleotides. Evidences point to their participation in Trypanosoma cruzi virulence and infectivity. In this work, we evaluate TcNTPDase-1 gene expression in comparison with ecto-NTPDase activity, in order to study the role of TcNTPDase-1 in parasite virulence, infectivity and adaptation to heat shock. Findings: Comparison between distinct T. cruzi isolates (Y, 3663 and 4167 strains, and Dm28c, LL014 and CL-14 clones) showed that TcNTPDase-1 expression was 7.2 ± 1.5 times higher in the Dm28c than the CL-14 avirulent clone. A remarkable expression increase was also observed in the trypomastigote and amastigote forms (22.5 ± 5.6 and 16.3 ± 3.8 times higher than epimastigotes, respectively), indicating that TcNTPDase-1 is overexpressed in T. cruzi infective forms. Moreover, heat shock and long-term cultivation also induced a significant increment on TcNTPDase-1 expression.

Research paper thumbnail of Diadenosine 5′,5′′-(Boranated)polyphosphonate Analogues as Selective Nucleotide Pyrophosphatase/Phosphodiesterase Inhibitors ⊥

Journal of Medicinal Chemistry, 2010

Nucleotide pyrophosphatase/phosphodiesterases (NPPs) hydrolyze extracellular nucleotides and dinu... more Nucleotide pyrophosphatase/phosphodiesterases (NPPs) hydrolyze extracellular nucleotides and dinucleotides and thus control purinergic signaling. Enhanced NPP activity is implicated in health disorders such as osteoarthritis and cancer. We designed novel diadenosine polyphosphonate derivatives as potential NPP inhibitors. Analogues 1-4 bear a phosphonate and/or boranophosphate group and/or a 2&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-H atom instead of a 2&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-OH group. In comparison to ATP, analogues 1-4 were barely hydrolyzed by human NTPDase1, -2, -3, and -8 (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;5% hydrolysis) and NPP1 and -3 (≤ 13%) and were not hydrolyzed by ecto-5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-nucleotidase, unlike AMP. These derivatives did not affect NTPDase activity, and analogues 1 and 2 did not inhibit ecto-5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-nucleotidase. All analogues blocked ∼80% of the NPP2-dependent hydrolysis of pnp-TMP, a specific NPP substrate, and inhibited the catabolism of pnp-TMP (K(i) and IC₅₀ both found to be between 10 and 60 μM), Ap₅A, and ATP by NPP1. The activity of NPP3 was inhibited to a lesser extent by the new analogues, with compounds 1 and 4 being the most effective in that respect. The analogues dramatically reduced the level of hydrolysis of pnp-TMP at the cell surface of both osteocarcinoma and colon cancer cells. Importantly, analogues 1-4 exhibited significantly reduced agonistic activity toward human P2Y₁,₁₁) receptors (except for analogue 1) and no activity with human P2Y₂ receptor. Our data provide strong evidence that analogue 2 is the first specific NPP inhibitor to be described.

Research paper thumbnail of Diadenosine and Diuridine Poly(borano)phosphate Analogues:  Synthesis, Chemical and Enzymatic Stability, and Activity at P2Y 1 and P2Y 2 Receptors †

Journal of Medicinal Chemistry, 2006

Dinucleoside polyphosphates, Np n N′, exert their physiological effects via P2 receptors. They ar... more Dinucleoside polyphosphates, Np n N′, exert their physiological effects via P2 receptors. They are attractive drug targets as they offer better stability and specificity compared to nucleotides, the most common P2receptor ligands. To further improve the properties of Np n N′, which are still pharmacologically unsatisfactory, we developed novel boranophosphate isosteres of dinucleoside polyphosphates, denoted as Np n (B)N. These analogues were obtained in a facile and efficient synthesis as the exclusive products in a concerted reaction of two nucleoside phosphorimidazolides and inorganic boranophosphate. This unusual reaction is due to the preorganization of three reactant molecules by the Mg 2+ ion. We found that Ap 3/5 ( /γ-B)A analogues were potent P2Y 1 -R agonists. Ap 5 (γ-B)A was equipotent to 2-MeS-ADP (EC 50 6.3 × 10 -8 M), thus making it one of the most potent P2Y 1 -R agonists currently known. Moreover, Ap 5 (γ-B)A did not activate P2Y 2 -R. In contrast, Up 3/5 ( /γ-B)U analogues were extremely poor agonists of both P2Y 1 -R and P2Y 2 -R. Np n (B)N analogues exhibited remarkable chemical stability under physiological conditions. Under conditions mimicking gastric juice, Np 3 ( -B)N analogues exhibited a half-life (t 1/2 ) of 1.3 h, whereas Np 5 (γ-B)N degraded at a much faster rate (t 1/2 18 min). The hydrolysis of Ap 3 ( -B)A by human nucleotide pyrophosphatase phosphodiesterases (NPP1 and NPP3) was slowed by 40% and 59%, respectively, as compared to Ap 3 A. However, this effect of the boranophosphate was position-dependent, as Np 5 (γ-B)N was degraded at a rate comparable to that of Np 5 N. In summary, Ap 5 (γ-B)A appears to be a highly potent and selective P2Y 1 -R agonist, as compared to the parent compound. This promising scaffold will be applied in the design of future metabolically stable analogues. † A U.S. patent was filed June 15, 2005.

Research paper thumbnail of Extracellular nucleotides mediate LPS-induced neutrophil migration in vitro and in vivo

Journal of Leukocyte Biology, 2007

Extracellular nucleotides are emerging as important inflammatory mediators. Here, we demonstrate ... more Extracellular nucleotides are emerging as important inflammatory mediators. Here, we demonstrate that these molecules mediate LPSinduced neutrophil migration in vitro and in vivo. Apyrase, a nucleotide scavenger, reduced the ability of LPS-stimulated monocytes to recruit neutrophils, as assayed using a modified Boyden chamber. This effect resulted from the inhibition of IL-8 release from monocytes. Furthermore, LPS-induced IL-8 release by monocytes was attenuated significantly by P2Y 6 receptor antagonists, RB-2 and MRS2578. Reciprocally, UDP, the selective P2Y 6 agonist, induced IL-8 release by monocytes. As for LPS, the media of UDP-stimulated monocytes were chemotactic for neutrophils; IL-8 accounted for ϳ50% of neutrophil migration induced by the media of LPS-or UDP-treated monocytes in transendothelial migration assays. It is important that in the murine air-pouch model, extracellular nucleotides were instrumental in LPSinduced neutrophil migration. Altogether, these data imply that LPS induces the release of nucleotides from monocytes and that by autocrine stimulation, the latter molecules regulate neutrophil migration caused by Gram-negative bacteria, suggesting a proinflammatory role of extracellular nucleotides in innate immunity. J. Leukoc. Biol. 81: 1269 -1275; 2007.

Research paper thumbnail of Nucleoside triphosphate diphosphohydrolase-2 is the ecto-ATPase of type I cells in taste buds

The Journal of Comparative Neurology, 2006