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

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 NAD_e ⁺-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 NAD_e ⁺ 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 NAD_e ⁺-induced intracellular increases of [Ca²⁺]_i and chemotaxis. These results demonstrate that β-NAD_e ⁺ 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.