Lysophosphatidic acid triggers calcium entry through a non-store-operated pathway in human neutrophils

The neuropeptide pituitary adenylate cyclase-activating protein (PACAP) acts via the G protein-coupled receptor vasoactive intestinal peptide/PACAP receptor-1 to induce phospholipase C/calcium and MAPK-dependent proinflammatory activities in human polymorphonuclear neutrophils (PMNs). In this study, we evaluate other mechanisms that regulate PACAP-evoked calcium transients, the nature of the calcium sources, and the role of calcium in proinflammatory activities. Reduction in the activity of PMNs to respond to PACAP was observed after cell exposure to inhibitors of the cAMP/protein kinase A, protein kinase C, and PI3K pathways, to pertussis toxin, genistein, and after chelation of intracellular calcium or after extracellular calcium depletion. Mobilization of intracellular calcium stores was based on the fact that PACAP-associated calcium transient was decreased after exposure to 1) thapsigargin, 2) Xestospongin C, and 3) the protonophore carbonyl cyanide 4-(trifluoromethoxy) phenyl hydrazone; inhibition of calcium increase by calcium channel blockers, by nifedipine and verapamil, indicated that PACAP was also acting on calcium influx. Such mobilization was not dependent on a functional actin cytoskeleton. Homologous desensitization with nanomoles of PACAP concentration and heterologous receptors desensibilization by G protein-coupled receptor agonists were observed. Intracellular calcium depletion modulated PACAP-associated ERK but not p38 phosphorylation; in contrast, extracellular calcium depletion modulated PACAP-associated p38 but not ERK phosphorylation. In PACAP-treated PMNs, reactive oxygen species production and CD11b membrane up-regulation in contrast to lactoferrin release were dependent on both intra- and extracellular calcium, whereas matrix metalloproteinase-9 release was unaffected by extracellular calcium depletion. These data indicate that both extracellular and intracellular calcium play key roles in PACAP proinflammatory activities.

Section: Discussionmentioning

confidence: 99%

Differential Calcium Regulation of Proinflammatory Activities in Human Neutrophils Exposed to the Neuropeptide Pituitary Adenylate Cyclase-Activating Protein

Harfi

Corazza

D’Hondt

et al. 2005

“…1A; C18:1/OH shown). As such, loss of integrity of the plasma membrane was the likely mechanism of the demonstrated prolonged calcium flux, and is reminiscent of published receptor-independent responses of neutrophils to lysophosphatidic acid (lyso-PA) and sphingosine 1-P, added either in water or methanol, respectively (14,22).…”

Section: Calcium Mobilization By Lyso-pc Depends On Mode Of Presentatmentioning

confidence: 99%

Lysophospholipids of Different Classes Mobilize Neutrophil Secretory Vesicles and Induce Redundant Signaling through G2A

Frasch

Berry²,

Murphy³

et al. 2007

Lysophosphatidylcholine has been shown to enhance neutrophil functions through a mechanism involving the G protein-coupled receptor G2A. Recent data support an indirect effect of lysophosphatidylcholine on G2A rather than direct ligand binding. These observations prompted the hypothesis that other lysophospholipids (lyso-PLs) may also signal for human neutrophil activation through G2A. To this end, 1-oleoyl-2-hydroxy-sn-glycero-3-[phospho-l-choline], but also C18:1/OH lyso-PLs bearing the phosphoserine and phosphoethanolamine head groups, presented on albumin, were shown to signal for calcium flux in a self- and cross-desensitizing manner, implicating a single receptor. Blocking Abs to G2A inhibited calcium signaling by all three lyso-PLs. Furthermore, inhibition by both pertussis toxin and U-73122 established signaling via the Gαi/phospholipase C pathway for calcium mobilization. Altered plasma membrane localization of G2A has been hypothesized to facilitate signaling. Accordingly, an increase in detectable G2A was demonstrated by 1 min after lyso-PL stimulation and was followed by visible patching of the receptor. Western blotting showed that G2A resides in the plasma membrane/secretory vesicle fraction and not in neutrophil primary, secondary, or tertiary granules. Enhanced detection of G2A induced by lyso-PLs was paralleled by enhanced detection of CD45, confirming mobilization of the labile secretory vesicle pool. Together, these data show that lyso-PLs bearing various head groups redundantly mobilize G2A latent within secretory vesicles and result in G2A receptor/Gαi/phospholipase C signaling for calcium flux in neutrophils.

“…These data support the fact that extracellular Ca 2+ entry regulates phagosome-induced ROS production. To obtain further evidence about the contribution of Ca 2+ influx in phagosomal oxidative activity, we incubated dHL-60 cells with La 3+ (30 mM) or SK&F-96365 (30 mM), two inhibitors of extracellular Ca 2+ entry, before the addition of labeled zymosan (20,26). Because the rate of ROS production is also strongly decreased by cell pretreatment using these inhibitors, we conclude that the regulation of phagosomal oxidative activity is dependent on extracellular Ca 2+ entry (Fig.…”

Section: Extracellular Ca 2+ Entry Regulates Phagosomal Oxidative Actmentioning

confidence: 99%

An Essential Role of STIM1, Orai1, and S100A8–A9 Proteins for Ca2+ Signaling and FcγR-Mediated Phagosomal Oxidative Activity

Steinckwich

Schenten

Melchior

et al. 2011

Phagocytosis is a process of innate immunity that allows for the enclosure of pathogens within the phagosome and their subsequent destruction through the production of reactive oxygen species (ROS). Although these processes have been associated with increases of intracellular Ca2+ concentrations, the mechanisms by which Ca2+ could regulate the different phases of phagocytosis remain unknown. The aim of this study was to investigate the Ca2+ signaling pathways involved in the regulation of FcγRs-induced phagocytosis. Our work focuses on IgG-opsonized zymosan internalization and phagosomal ROS production in DMSO-differentiated HL-60 cells and neutrophils. We found that chelation of intracellular Ca2+ by BAPTA or emptying of the intracellular Ca2+ store by thapsigargin reduced the efficiency of zymosan internalization. Using an small interfering RNA strategy, our data establish that the observed Ca2+ release occurs through two isoforms of inositol 1,4,5-triphosphate receptors, ITPR1 and ITPR3. In addition, we provide evidence that phagosomal ROS production is dependent on extracellular Ca2+ entry. We demonstrate that the observed Ca2+ influx is supported by ORAI calcium release-activated calcium modulator 1 (Orai1) and stromal interaction molecule 1 (STIM1). This result suggests that extracellular Ca2+ entry, which is required for ROS production, is mediated by a store-operated Ca2+ mechanism. Finally, our data identify the complex formed by S100A8 and S100A9 (S100 calcium-binding protein A8 and A9 complex), two Ca2+-binding proteins, as the site of interplay between extracellular Ca2+ entry and intraphagosomal ROS production. Thus, we demonstrate that FcγR-mediated phagocytosis requires intracellular Ca2+ store depletion for the internalization phase. Then phagosomal ROS production requires extracellular Ca2+ entry mediated by Orai1/STIM1 and relayed by S100A8–A9 as Ca2+ sensor.