Sequencing, functional expression and characterization of rat NTPDase6, a nucleoside diphosphatase and novel member of the ecto-nucleoside triphosphate diphosphohydrolase family

Braun, Norbert; Fengler, Sabine; Ebeling, Christian; Servos, Jörg; Zimmermann, Herbert

doi:10.1042/bj3510639

Cited by 52 publications

(10 citation statements)

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“…The biochemistry of the NTPDase1 catalytic activity is well described for the pancreatic tissue (see Introduction), but our observation that a similar hydrolytic activity is found in pancreatic juice is new. Recently, several reports show that some nucleotidases (NTPDases 5 and 6; and NPP1 to NPP3) may be released into the surrounding medium of various cells (Mulero et al 1999; Bollen et al 2000; Braun et al 2000; Chen & Guidotti, 2001). Also sympathetic nerve stimulation causes release of soluble NTPDase along with ATP (Todorov et al 1997; Westfall et al 2000).…”

Section: Resultsmentioning

confidence: 99%

“…NTPDases 5 and 6 have the N‐terminal in the membrane, while the rest of the molecule is extra‐membranous and can be cleaved to yield a soluble protein. NTPDases 1‐3 are bound to the plasma membranes, while NTPDases 4‐6 can be associated with intracellular organelles (Braun et al 2000; Zimmermann, 2000). Another family of enzymes, ecto‐nucleotide pyrophosphatase/ phosphodiesterases (NPP), has broad substrate specificity.…”

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confidence: 99%

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Rat pancreas secretes particulate ecto-nucleotidase CD39

et al. 2003

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In exocrine pancreas, acini release ATP and the excurrent ducts express several types of purinergic P2 receptors. Thereby, ATP, or its hydrolytic products, might play a role as a paracrine regulator between acini and ducts. The aim of the present study was to elucidate whether this acinar-ductal signalling is regulated by nucleotidase(s), and to characterize and localize one of the nucleotidases within the rat pancreas. Using RT-PCR and Western blotting we show that pancreas expresses the full length ecto-nucleoside triphosphate diphosphohydrolase, CD39. Immunofluorescence shows CD39 localization on basolateral membranes of acini and intracellularly. In small intercalated/ interlobular ducts, CD39 immunofluorescence was localized on the luminal membranes, while in larger ducts it was localized on the basolateral membranes. Upon stimulation with cholecystokininoctapeptide-8 (CCK-8), acinar CD39 relocalizes in clusters towards the lumen and is secreted. As a result, pancreatic juice collected from intact pancreas stimulated with CCK-8 contained nucleotidase activity, including that of CD39, and no detectable amounts of ATP. Anti-CD39 antibodies detected the full length (78 kDa) CD39 in pancreatic juice. This CD39 was confined only to the particulate and not to the soluble fraction of CCK-8-stimulated secretion. No CD39 activity was detected in secretion stimulated by secretin. The role of secreted particulate, possibly microsomal, CD39 would be to regulate intraluminal ATP concentrations within the ductal tree. In conclusion, we show a novel inducible release of full length particulate CD39, and propose its role in the physiological context of pancreatic secretion.

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Section: Resultsmentioning

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Rat pancreas secretes particulate ecto-nucleotidase CD39

et al. 2003

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“…NTPDase5 and NTPDase6 function primarily as nucleoside diphosphatases (Mulero et al . 1999; Braun et al . 2000a; Hicks‐Berger et al .…”

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Multiple ecto‐nucleotidases in PC12 cells: identification and cellular distribution after heterologous expression

Vollmayer¹,

Koch²,

Braun

et al. 2001

Journal of Neurochemistry

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The physiological action of extracellular ATP and other nucleotides in the nervous system is controlled by surfacelocated enzymes (ecto-nucleotidases) of which several families with partially overlapping substrate speci®cities exist. In order to identify ecto-nucleotidases potentially associated with neural cells, we chose PC12 cells for analysis. PC12 cells revealed surface-located ATPase and ADPase activity with apparent K m -values of 283 mM and 243 mM, respectively. Using PCR we identi®ed the mRNA of all members of the ecto-nucleoside triphosphate diphosphohydrolase family investigated (NTPDase1 to NTPDase3, NTPDase5/6), of ecto-nucleotide pyrophosphatase/phosphodiesterase3 (NPP3), tissue-non-speci®c alkaline phosphatase and ecto-5 H -nucleotidase. The surface-located catalytic activity differed greatly between the various enzyme species. Our data suggest that hydrolysis of ATP and ADP is mainly due to members of the ecto-nucleoside triphosphate diphosphohydrolase family. Activity of ecto-5 H -nucleotidase and alkaline phosphatase was very low and activity of NPP3 was absent.For a detailed analysis of the cellular distribution of ectonucleotidases single and double transfections of PC12 cells were performed, followed by¯uorescence analysis. Ectonucleotidases were distributed over the entire cell surface and accumulated intracellularly in varicosities and neurite tips. PC12 cell ecto-nucleotidases are likely to play an important role in terminating autocrine functions of released nucleotides and in producing extracellular nucleosides supporting the survival and neuritic differentiation of PC12 cells.

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“…Both enzymes are located intracellularly, but have intra- and extracellular functionality as they can be released from cells. NTPDase5 and 6 have high preference for nucleoside 50-diphosphates, such as UDP and GDP ( 5 , 6 ).…”

Section: Communicationsmentioning

confidence: 99%

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Montopoli¹,

Ragazzi²,

Caparrotta³

et al. 2006

Purinergic Signalling

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Following a brief account of the early history of the discovery of purinergic signalling, a personal view of some of the exciting cutting-edge directions being taken by research in the field will be considered. In particular, emphasis will be placed on the pathophysiology of purinergic signalling and its therapeutic potential.Recent advances in understanding the role of adenosine and its receptors in physiology and pathophysiology as well as new developments in medicinal chemistry of these receptors have made it possible to begin to realize the therapeutic potential of adenosine and its receptors. Currently adenosine itself is targeted at the heart; a bolus of adenosine intravenously is commonly used to treat supraventricular tachycardia whereas an infusion of adenosine is used as a coronary vasodilator during pharmacologic stress testing. Non-selective adenosine receptor antagonists are used to maintain wakefulness (caffeine), as an analgesic (caffeine) and, less commonly at present, to treat bronchospasm (theophylline, aminophylline, enprofylline). Currently a number of new selective adenosine receptor agonists and antagonists are in testing for a variety of new indications and one adenosinetargeted indication. The older indication is pharmacologic stress testing and new selective A 2A receptor agonists, ATL146e (Apadenoson, Adenosine Therapeutics) and Regadenoson (CV Therapeutics), are currently under study (Phase II-III) for this indication. In addition, a selective A 1 receptor agonist is in trials for supraventricular tachycardia (tecadenoson, CV Therapeutics), Currently a number of companies are testing selective adenosine receptor agonists for newer indications including Rheumatoid Arthritis, cancer and wound healing, Adenosine A 3 receptor agonists are in trials for the treatment of Rheumatoid Arthritis and Cancer (CF101, CF102, Can-Fite Biopharmaceuticals) and preliminary promising results in patients with RA have been reported. Topical application of an adenosine A 2A receptor agonist to promote healing of diabetic foot ulcers is currently in Phase II trials after the successful completion of a Phase I safety/early efficacy trial (MRE94, King Pharmaceuticals). An allosteric enhancer at the adenosine A 1 receptor is in trials for neuropathic pain (T-62, King Pharmaceuticals). Because adenosine receptor stimulation may also be involved in disease pathogenesis selective adenosine receptor antagonists are being studied for select indications as well. Istradefylline (KW-6002, Kyowa Pharmaceuticals), a selective adenosine A 2A receptor antagonist, is now in late stage clinical trials for the treatment of Parkinson"s Disease. Another approach to targeting adenosine receptors is to increase extracellular adenosine concentrations, mechanisms shown to mediate the anti-inflammatory effects of two drugs commonly used in the treatment of Rheumatoid Arthritis, methotrexate and sulfasalazine. Dipyridamole inhibits adenosine uptake and this clearly underlies its effect as a coronary vasodilator during pharmacologic ca...

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Sequencing, functional expression and characterization of rat NTPDase6, a nucleoside diphosphatase and novel member of the ecto-nucleoside triphosphate diphosphohydrolase family

Cited by 52 publications

References 36 publications

Rat pancreas secretes particulate ecto-nucleotidase CD39

Rat pancreas secretes particulate ecto-nucleotidase CD39

Multiple ecto‐nucleotidases in PC12 cells: identification and cellular distribution after heterologous expression

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