Extracellular NAD+ is an agonist of the human P2Y11 purinergic receptor in human granulocytes.
Micromolar concentrations of extracellular beta-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 (IP3) and cAMP, with a two-step elevation of intracellular calcium levels ([Ca2+]i): a rapid, IP3-mediated Ca2+ release, followed by a sustained influx of extracellular Ca2+ mediated by cADPR. Suramin, an inhibitor of P2Y receptors, abrogated NAD(e)+-induced intracellular increases of IP3, cAMP, cADPR, and [Ca2+]i, suggesting a role for a P2Y receptor coupled to both phospholipase C and adenylyl cyclase. The P2Y(11) receptor is the only known member of the P2Y receptor subfamily coupled to both phospholipase C and adenylyl cyclase. Therefore, we performed experiments on hP2Y(11)-transfected 1321N1 astrocytoma cells: micromolar NAD(e)+ promoted a two-step elevation of the [Ca2+]i due to the enhanced intracellular production of IP3, cAMP, and cADPR in 1321N1-hP2Y(11) but not in untransfected 1321N1 cells. In human granulocytes NF157, a selective and potent inhibitor of P2Y(11), and the down-regulation of P2Y(11) expression by short interference RNA prevented NAD(e)+-induced intracellular increases of [Ca2+]i and chemotaxis. These results demonstrate that beta-NAD(e)+ is an agonist of the P2Y(11) purinoceptor and that P2Y(11) is the endogenous receptor in granulocytes mediating the sustained [Ca2+]i increase responsible for their functional activation.
Moreschi
Iliana
I
Department of Experimental Medicine, Section of Biochemistry, and Center of Excellence for Biomedical Research (CEBR), University of Genova, Viale Benedetto XV/1, 16132 Genova, Italy.
Bruzzone
Santina
S
Nicholas
Robert A
RA
Fruscione
Floriana
F
Sturla
Laura
L
Benvenuto
Federica
F
Usai
Cesare
C
Meis
Sabine
S
Kassack
Matthias U
MU
Zocchi
Elena
E
De Flora
Antonio
A
D045744:Cell Line, Tumor; D002633:Chemotaxis; D036563:Cyclic ADP-Ribose / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D015536:Down-Regulation; D006098:Granulocytes / Q000378:metabolism*; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D009243:NAD / Q000737:chemistry*; D058910:Purinergic P2 Receptor Agonists; D034741:RNA, Small Interfering / Q000378:metabolism; D018048:Receptors, Purinergic P2; D014162:Transfection
C109734:P2RY11 protein, human; D058910:Purinergic P2 Receptor Agonists; D034741:RNA, Small Interfering; D018048:Receptors, Purinergic P2; D009243:NAD; D036563:Cyclic ADP-Ribose; D015544:Inositol 1,4,5-Trisphosphate; D000242:Cyclic AMP