Cyclic ADP-Ribose Mediates Formyl Methionyl Leucyl Phenylalanine (fMLP)-Induced Intracellular Ca2+Rise and Migration of Human Neutrophils

Abstract

Although cyclic ADP-ribose (cADPR), a novel Ca2+-mobilizing mediator, is suggested to be involved in the functions of neutrophils in rodents, its role in human neutrophils remains unclear. The present study examined the ability of cADPR to mobilize Ca2+and mediate formyl methionyl leucyl phenylalanine (fMLP)-stimulated increase in cytosolic free Ca2+concentration ([Ca2+]i) and migration in human neutrophils. cADPR induced Ca2+release from digitonin-permeabilized neutrophils, and the release was blocked by 8Br-cADPR, an antagonist of cADPR. Immunophilin ligands, FK506 and rapamycin, but not cyclosporine A, inhibited cADPR-induced Ca2+release. 8Br-cADPR partially reduced fMLP-induced [Ca2+]irise and abolished the rise in combination with 2APB, an IP3-receptor antagonist. Anti-CD38Ab and NADase that interfere with cADPR formation, reduced the fMLP-induced [Ca2+]irise. When β-NAD+, a substrate of ADP-ribosyl cyclase, and cADPR were added to the medium, the former gradually increased [Ca2+]iand the latter potentiated the fMLP-induced [Ca2+]irise. The β-NAD+–induced [Ca2+]irise in Ca2+-free medium was inhibited by anti-CD38Ab, 8Br-cADPR, FK506, ruthenium red, and thapsigargin. mRNAs of nucleoside transporter (NT), ENT1, ENT2, CNT, and CNT3 were expressed in neutrophils; and their inhibitors, inosine, uridine, and s-(4-nitrobenzyl)-6-thioinosine, reduced the [Ca2+]irise induced by β-NAD+and fMLP. fMLP-stimulated migration was inhibited by the removal of Ca2+from the medium or by the addition of 8Br-cADPR, anti-CD38Ab, NADase, and NT inhibitors. These results suggest that cADPR was synthesized extracellularly by CD38, transported into the cells through NTs, and then Ca2+was mobilized by FK506-binding protein–dependent process. This process may be involved in fMLP-induced intracellular Ca2+signaling and migration in human neutrophils. Keywords:: cyclic ADP-ribose (cADPR), FK506-binding protein (FKBP), cytosolic Ca2+dynamics, nucleoside transporter, migration