Molecular mechanism of nucleotide-induced primary granule release in human neutrophils: role for the P2Y₂ receptor

Abstract

otides are released during vascular injury from activated platelets and broken cells, which could stimulate human neutrophils. In this study, we characterized the P2Y receptors and investigated the functional effects of extracellular nucleotides on human neutrophils. Pharmacological characterization using selective agonists and pertussis toxin revealed that human neutrophils express only functional P2Y 2 receptors. However, P2Y 2 receptor agonists ATP or uridine triphosphate (UTP) caused intracellular Ca 2ϩ increases in isolated human neutrophils with an EC 50 of 1 M but failed to cause release of primary granules from human neutrophils. ATP and UTP were equally potent in causing elastase release from human neutrophils in the presence of exogenous soluble fibrinogen, whereas ADP and UDP were without effect. We investigated whether nucleotides depend on generated arachidonic acid metabolites to cause degranulation. However, phenidone and MK-886, inhibitors of the 5-lipoxygenase pathway, failed to block nucleotide-induced intracellular calcium mobilization and elastase release. ATP and UTP caused activation of p38 MAPK and ERK1/2 in human neutrophils. In addition, the inhibitors of the MAPK pathway, SB-203580 and U-0126, inhibited nucleotide-induced elastase release. We conclude that fibrinogen is required for nucleotide-induced primary granule release from human neutrophils through the P2Y 2 receptor without a role for arachidonic acid metabolites. Both ERK1/2 and p38 MAPK play an important role in nucleotide-induced primary granule release from human neutrophils.

GTP-dependent Secretion from Neutrophils Is Regulated by Cdk5

“…Several proteins have been implicated in regulating secretion from neutrophils, including the small ras-related GTPases (3,4), annexins (5), and the mitogen-activated protein kinases (6). Activation of both p38 mitogen-activated protein kinase and extracellular signal-regulated kinase 1/2 has also been demonstrated in ATP-and UTP-stimulated neutrophils (7), supporting an important role for kinases in nucleotide-induced secretion. In this study, we begin to identify the protein effectors of the GTP-mediated secretory machinery in neutrophils.…”

Extracellular nucleotides mediate LPS-induced neutrophil migration in vitro and in vivo

“…Primary monocytes and HUVEC express the mRNA of P2Y 1 , P2Y 2 , P2Y 4 , and P2Y 6 receptors, and polymorphonuclear leukocytes appears to express all P2Y receptor mRNAs except P2Y 12 [4][5][6]. In neutrophils, P2Y 2 appeared dominantly expressed at the mRNA level and was also functionally demonstrated at the protein level [5,7,8]. P2Y 2 and P2Y 11 are activated by ATP, P2Y 1 and P2Y 13 by ADP, P2Y 4 by UTP, P2Y 6 by UDP, and P2Y 14 by UDP glucose [1].…”

NTPDase1 Controls IL-8 Production by Human Neutrophils

“…All P2X and P2Y 11 receptors are activated by ATP; P2Y 2 receptors by ATP and UTP; P2Y 1 and P2Y 13 receptors by ADP; P2Y 4 receptors by UTP; P2Y 6 receptors by UDP; and P2Y 14 receptors by UDP-glucose (6). To date, most of the key inflammatory responses that are regulated by ATP in human neutrophils appear to involve the P2Y 2 receptor, which is thought to be the main species expressed at the mRNA and protein levels (5,7). P2Y 2 has been implicated in neutrophil chemotaxis (5,8,9), arachidonic acid release (10), degranulation, and oxidative burst (11)(12)(13).…”