NAADP+ is an agonist of the human P2Y11 purinergic receptor

Nicotinic acid adenine dinucleotide phosphate (NAADP+) is an intracellular second messenger releasing Ca2+ from intracellular stores in different cell types. In addition, it is also active in triggering [Ca2+]i increase when applied extracellularly and various underlying mechanisms have been proposed. Here, we used hP2Y11-transfected 1321N1 astrocytoma cells to unequivocally establish whether extracellular NAADP+ is an agonist of the P2Y11 receptor, as previously reported for β-NAD+ [I. Moreschi, S. Bruzzone, R.A. Nicholas, et al., Extracellular NAD+ is an agonist of the human P2Y11 purinergic receptor in human granulocytes, J. Biol. Chem. 281 (2006) 31419–31429]. Extracellular NAADP+ triggered a concentration-dependent two-step elevation of [Ca2+]i in 1321N1-hP2Y11 cells, but not in wild-type 1321N1 cells, secondary to the intracellular production of IP3, cAMP and cyclic ADP-ribose (cADPR). Specifically, the transient [Ca2+]i rise proved to be related to IP3 overproduction and to consequent Ca2+ mobilization, while the sustained [Ca2+]i elevation was caused by the cAMP/ADP-ribosyl cyclase (ADPRC)/cADPR signalling cascade and by influx of extracellular Ca2+. In human granulocytes, endogenous P2Y11 proved to be responsible for the NAADP+-induced cell activation (as demonstrated by the use of NF157, a selective and potent inhibitor of P2Y11), unveiling a role of NAADP+ as a pro-inflammatory cytokine. In conclusion, we provide unequivocal evidence for the activation of a member of the P2Y receptor subfamily by NAADP+.