A Selective High-Affinity Antagonist of the P2Y₁₄ Receptor Inhibits UDP-Glucose–Stimulated Chemotaxis of Human Neutrophils

Abstract

The nucleotide-sugar-activated P2Y 14 receptor (P2Y 14 -R) is highly expressed in hematopoietic cells. Although the physiologic functions of this receptor remain undefined, it has been strongly implicated recently in immune and inflammatory responses. Lack of availability of receptor-selective high-affinity antagonists has impeded progress in studies of this and most of the eight nucleotide-activated P2Y receptors. A series of molecules recently were identified by Gauthier et al. ) that exhibited antagonist activity at the P2Y 14 -R. We synthesized one of these molecules, a 4,7-disubstituted 2-naphthoic acid derivative (PPTN), and studied its pharmacological properties in detail. The concentration-effect curve of UDP-glucose for promoting inhibition of adenylyl cyclase in C6 glioma cells stably expressing the P2Y 14 -R was shifted to the right in a concentration-dependent manner by PPTN.Schild analyses revealed that PPTN-mediated inhibition followed competitive kinetics, with a K B of 434 pM observed. In contrast, 1 mM PPTN exhibited no agonist or antagonist effect at the P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , or P2Y 13 receptors. UDP-glucose-promoted chemotaxis of differentiated HL-60 human promyelocytic leukemia cells was blocked by PPTN with a concentration dependence consistent with the K B determined with recombinant P2Y 14 -R. In contrast, the chemotactic response evoked by the chemoattractant peptide fMetLeuPhe was unaffected by PPTN. UDP-glucose-promoted chemotaxis of freshly isolated human neutrophils also was blocked by PPTN. In summary, this work establishes PPTN as a highly selective high-affinity antagonist of the P2Y 14 -R that is useful for interrogating the action of this receptor in physiologic systems.

UDP-glucose promotes neutrophil recruitment in the lung

“…PPTN acts as a high-affinity competitive antagonist of the P2Y 14 R and does so without interacting with any of the other seven P2Y receptor subtypes [30]. Furthermore, PPTN blocks UDPglucose-promoted chemotaxis of human neutrophils without affecting responses to bacterial peptides [30].…”

“…We recently illustrated that the P2Y 14 R is functionally expressed in freshly isolated blood neutrophils, promoting cytoskeleton rearrangement, Rho activation, and cell migration [29,30]. Therefore, an additional mechanism likely contributing to UDP-glucose-promoted neutrophil lung inflammation is via activation of the neutrophil P2Y 14 R. For example, UDP-glucose released into ASL could diffuse through the paracellular pathway generating trans-epithelial and transendothelial gradients that direct neutrophils from the vasculature to the airways.…”

“…We recently reported that the naphthoic acid derivative PPTN is a high-affinity competitive antagonist of the P2Y 14 R that does not interact with any of the other seven P2Y receptor subtypes [30]. UDP-glucose-stimulated neutrophil lung infiltration was almost completely blocked when the UDP-sugar was co-instilled with PPTN (Fig.…”

“…PPTN was synthesized as previously described [30] and used as a dimethylsulfoxide (DMSO) stock solution (100 mM), which was stored at −20°C. PPTN was freshly diluted in endotoxinfree PBS before its administration to mice and an equivalent dilution of DMSO in PBS was used as vehicle in control animals.…”

“…In addition, we recently demonstrated that UDP-glucose promotes P2Y 14 R-mediated chemotaxis of freshly isolated human neutrophils. Enhanced PMN migration was observed in response to UDP-glucose gradients, and this activity was abolished when the P2Y 14 R antagonist PPTN [4-(piperidin-4-yl)-phenyl)-7-(4-(trifluoromethyl)-phenyl-2-naphthoic acid] was included in the cell suspension buffer [29,30]. We also showed that UDP-glucose-promoted chemotaxis in neutrophil-like HL60 cells was absolutely dependent on the expression of P2Y 14 R in these cells and was abolished by PPTN [29,30].…”