Extracellular NAD+ is an agonist of the human P2Y11 purinergic receptor in human granulocytes

Iliana Moreschi, Santina Bruzzone, Robert A. Nicholas, Floriana Fruscione, Laura Sturla, Federica Benvenuto, Cesare Usai, Sabine Meis, Matthias U. Kassack, Elena Zocchi, Antonio De Flora

Research output: Contribution to journalArticlepeer-review

Abstract

Micromolar concentrations of extracellular β-NAD+ (NAD e +) activate human granulocytes (superoxide and NO generation and chemotaxis) by triggering: (i) overproduction of cAMP, (ii) activation of protein kinase A, (iii) stimulation of ADP-ribosyl cyclase and overproduction of cyclic ADP-ribose (cADPR), a universal Ca2+ mobilizer, and (iv) influx of extracellular Ca2+. Here we demonstrate that exposure of granulocytes to millimolar rather than to micromolar NAD e + generates both inositol 1,4,5-trisphosphate (IP 3) and cAMP, with a two-step elevation of intracellular calcium levels ([Ca2+]i): a rapid, IP3-mediated Ca 2+ release, followed by a sustained influx of extracellular Ca 2+ mediated by cADPR. Suramin, an inhibitor of P2Y receptors, abrogated NADe +-induced intracellular increases of IP 3, cAMP, cADPR, and [Ca2+]i, suggesting a role for a P2Y receptor coupled to both phospholipase C and adenylyl cyclase. The P2Y11 receptor is the only known member of the P2Y receptor subfamily coupled to both phospholipase C and adenylyl cyclase. Therefore, we performed experiments on hP2Y11-transfected 1321N1 astrocytoma cells: micromolar NADe + promoted a two-step elevation of the [Ca2+]i due to the enhanced intracellular production of IP3, cAMP, and cADPR in 1321N1-hP2Y11 but not in untransfected 1321N1 cells. In human granulocytes NF157, a selective and potent inhibitor of P2Y11, and the down-regulation of P2Y11 expression by short interference RNA prevented NADe +-induced intracellular increases of [Ca2+]i and chemotaxis. These results demonstrate that β-NADe + is an agonist of the P2Y11 purinoceptor and that P2Y11 is the endogenous receptor in granulocytes mediating the sustained [Ca2+]i increase responsible for their functional activation.

Original languageEnglish
Pages (from-to)31419-31429
Number of pages11
JournalJournal of Biological Chemistry
Volume281
Issue number42
DOIs
Publication statusPublished - Oct 20 2006

ASJC Scopus subject areas

  • Biochemistry

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