Signaling pathways downstream of P2 receptors in human neutrophils

Extracellular nucleotides stimulate human neutrophils by activating the purinergic P2Y2 receptor. However, it is not completely understood which types of G proteins are activated downstream of this P2 receptor subtype. We investigated the G-protein coupling to P2Y2 receptors and several subsequent signaling events. Treatment of neutrophils with pertussis toxin (PTX), a Gi protein inhibitor, caused only ∼75% loss of nucleotide-induced Ca2+ mobilization indicating that nucleotides cause Ca2+ mobilization both through Gi-dependent and Gi-independent pathways. However, the PLC inhibitor U73122 almost completely inhibited Ca2+ mobilization in both nucleotide- and fMLP-stimulated neutrophils, strongly supporting the view that both the PTX-sensitive and the PTX-insensitive mechanism of Ca2+ increase require activation of PLC. We investigated the dependence of ERK phosphorylation on the Gi pathway. Treatment of neutrophils with PTX caused almost complete inhibition of ERK phosphorylation in nucleotide or fMLP activated neutrophils. U73122 caused inhibition of nucleotide- or fMLP-stimulated ERK phosphorylation, suggesting that although pertussis toxin-insensitive pathways cause measurable Ca2+ mobilization, they are not sufficient for causing ERK phosphorylation. Since PLC activation leads to intracellular Ca2+ increase and PKC activation, we investigated if these intracellular events are necessary for ERK phosphorylation. Exposure of cells to the Ca2+ chelator BAPTA had no effect on nucleotide- or fMLP-induced ERK phosphorylation. However, the PKC inhibitor GF109203X was able to almost completely inhibit nucleotide- or fMLP-induced ERK phosphorylation. We conclude that the P2Y2 receptor can cause Ca2+ mobilization through a PTX-insensitive but PLC-dependent pathway and ERK phosphorylation is highly dependent on activation of the Gi proteins

The priming effect of extracellular UTP on human neutrophils: Role of calcium released from thapsigargin-sensitive intracellular stores

P2Y2 receptors, which are equally responsive to ATP and UTP, can trigger intracellular signaling events, such as intracellular calcium mobilization and mitogen-activated protein (MAP) kinase phosphorylation in polymorphonuclear leukocytes (PMN). Moreover, extracellular nucleotides have been shown to prime chemoattractant-induced superoxide production. The aim of our study was to investigate the mechanism responsible for the priming effect of extracellular nucleotides on reactive oxygen species (ROS) production induced in human neutrophils by two different chemoattractants: formyl-methionyl-leucyl-phenylalanine (fMLP) and interleukin-8 (IL-8). Nucleotide-induced priming of ROS production was concentration- and time-dependent. When UTP was added to neutrophil suspensions prior to chemoattractant, the increase of the response reached the maximum at 1 min of pre-incubation with the nucleotide. UTP potentiated the phosphorylation of p44/42 and p38 MAP kinases induced by chemoattractants, however the P2 receptor-mediated potentiation of ROS production was still detectable in the presence of a SB203580 or U0126, supporting the view that MAP kinases do not play a major role in regulating the nucleotide-induced effect. In the presence of thapsigargin, an inhibitor of the ubiquitous sarco-endoplasmic reticulum Ca2+-ATPases in mammalian cells, the effect of fMLP was not affected, but UTP-induced priming was abolished, suggesting that the release of calcium from thapsigargin-sensitive intracellular stores is essential for nucleotide-induced priming in human neutrophils

Substance P Induces Rapid and Transient Membrane Blebbing in U373MG Cells in a p21-Activated Kinase-Dependent Manner

U373MG astrocytoma cells endogenously express the full-length neurokinin 1 receptor (NK1R). Substance P (SP), the natural ligand for NK1R, triggers rapid and transient membrane blebbing and we report that these morphological changes have different dynamics and intracellular signaling as compared to the changes that we have previously described in HEK293-NK1R cells. In both cell lines, the SP-induced morphological changes are Gq-independent, and they require the Rho, Rho-associated coiled-coil kinase (ROCK) signaling pathway. Using confocal microscopy we have demonstrated that tubulin is phosphorylated subsequent to cell stimulation with SP and that tubulin accumulates inside the blebs. Colchicine, a tubulin polymerization inhibitor, blocked SP-induced blebbing in U373MG but not in HEK293-NK1R cells. Although p21-activated kinase (PAK) is expressed in both cell lines, SP induced rapid phosphorylation of PAK in U373MG, but failed to phosphorylate PAK in HEK293-NK1R cells. The cell-permeable Rho inhibitor C3 transferase inhibited SP-induced PAK phosphorylation, but the ROCK inhibitor Y27632 had no effect on PAK phosphorylation, suggesting that Rho activates PAK in a ROCK-independent manner. Our study demonstrates that SP triggers rapid changes in cell morphology mediated by distinct intracellular signaling mechanisms in U373MG versus HEK293-NK1R cells

Neurokinin 1 receptor mediates membrane blebbing and sheer stress-induced microparticle formation in HEK293 cells.

Cell-derived microparticles participate in intercellular communication similar to the classical messenger systems of small and macro-molecules that bind to specialized membrane receptors. Microparticles have been implicated in the regulation of a variety of complex physiopathologic processes, such as thrombosis, the control of innate and adaptive immunity, and cancer. The neurokinin 1 receptor (NK1R) is a Gq-coupled receptor present on the membrane of a variety of tissues, including neurons in the central and peripheral nervous system, immune cells, endocrine and exocrine glands, and smooth muscle. The endogenous agonist of NK1R is the undecapeptide substance P (SP). We have previously described intracellular signaling mechanisms that regulate NK1R-mediated rapid cell shape changes in HEK293 cells and U373MG cells. In the present study, we show that the activation of NK1R in HEK293 cells, but not in U373MG cells, leads to formation of sheer-stress induced microparticles that stain positive with the membrane-selective fluorescent dye FM 2-10. SP-induced microparticle formation is independent of elevated intracellular calcium concentrations and activation of NK1R present on HEK293-derived microparticles triggers detectable calcium increase in SP-induced microparticles. The ROCK inhibitor Y27632 and the dynamin inhibitor dynasore inhibited membrane blebbing and microparticle formation in HEK293 cells, strongly suggesting that microparticle formation in this cell type is dependent on membrane blebbing

SP triggers sheer-stress induced microparticle formation in HEK293-NK1R cells in a dose-dependent manner.

<p>HEK293-NK1R cell suspensions were incubated for 10 minutes with different concentrations of SP as indicated and then analyzed by flow cytometry. <i>Panels (A–D)</i> Density plots showing intact cells (P1) and microparticles (P2 and P3) detected based on their light scattering properties: forward scatter (FSC-A) and side scatter (SSC-A). <i>Panels E–H)</i> Dot plots showing the staining pattern with the cytosolic dye CFMDA (FL1-A) and Nuclear-ID Red stain (FL4-A) in intact cells and microparticles. <i>Panels I and J)</i> Representative histograms showing the distribution of CFMDA fluorescence intensities (FL1-A) for P2 and P3 microparticles. <i>Panel K)</i> Line graph showing mean values ± SEM (n = 4) of microparticle counts expressed as percent increase as compared to baseline-stimulated cells. The number of microparticles in the P2 population significantly increased with the concentration on SP (p<0.05). No significant increase in the number of microparticles in the P3 population was found.</p

SP induces detectable intracellular calcium increase in P2 microparticles, but not in P3 microparticles.

<p>HEK293-NK1R cells were loaded with fluo-4 and fura red, as described under <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045322#s2" target="_blank">Materials and Methods</a></i> and then stimulated with SP (100 nM) to generate microparticles. Microparticles and cells were washed to remove SP, and incubated for one hour at 37°C to allow NK1R to resensitize. SP (100 nM; panels A<i>–</i>H) was added to resensitized cell suspensions while performing intracellular calcium recordings. Aprepitant (1 µM) was used to block NK1R before addition of SP (panels E–H). ATP (1 µM; panels I<i>–</i>L), a P2 receptor agonist, and ionomycin (1 µM; panels M<i>–</i>P) were used to trigger intracellular calcium response independent of NK1R activation. <i>Panels A, E, I, M</i> show light scattering properties of representative samples. Line graphs of mean ratio (fluo-4/fura red) vs. time overlapped on pseudocolored dot plots ratio vs. time are shown in the remaining panels. <i>Panels A–D)</i> SP induced obvious intracellular calcium increase in intact cells and P2 microparticles, while P3 microparticles did not respond with detectable calcium increase to SP. <i>Panels E–H)</i> The NK1R antagonist aprepitant completely blocked the effect of SP, indicating that calcium increase is due to NK1R activation in intact cells as well as in P2 microparticles. Calcium increase induced by ATP (<i>Panels I–L)</i> and ionomycin (<i>Panels M–P</i>) are shown for comparison.</p

P2 microparticles contain NK1R.

<p>HEK293 cells were transfected with a plasmid encoding the NK1R-GFP fusion protein. HEK293-NK1R-GFP and HEK293-NK1R cells were stimulated with SP to generate microparticles, then analyzed by flow cytometry. <i>Upper panels:</i> histograms showing the distribution of GFP fluorescence in transfected cells, as compared to control cells. <i>Lower panels:</i> median fluorescence intensities obtained from three experiments. Intact cells (<i>upper panel A</i>), P2 (<i>upper panel B</i>) and P3 (<i>upper panel C</i>) microparticles exhibited higher fluorescence levels in samples transfected with GFP than in control samples transfected with untagged NK1R. Lower panels show median fluorescence intensities obtained from three experiments (mean values ± SEM; *p<0.05).</p

P2 and P3 microparticles originate from the plasma membrane.

<p><i>Panels A–C)</i> Microparticles were generated by SP addition to HEK293-NK1R cells and stained with the plasma membrane dye FM 2-10 as described under <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045322#s2" target="_blank">Materials and Methods</a></i>. Intact cells (A), P2 (B) and P3 (C) microparticles all stained positive, indicating that these vesicles contain membrane. <i>Upper panels</i> (grey curves: control cells or microparticles; red curves: stained cells or microparticles) and bar graphs (lower panels) showing median fluorescence values (MFI) for unstained and stained samples. A bimodal distribution histogram was found for the P3 population of microparticles (upper panel C) and the median fluorescence intensity was calculated separately for the dim and bright subpopulations of P3 microparticles (lower panel C). Data are derived from three independent experiments (mean values ± SEM; *p<0.05). <i>Panels D–F)</i> Microparticles were centrifuged at 10,000 g for 10 minutes. Both P2 and P3 microparticles were removed by centrifugation, indicating that these are not exosomes or protein aggregates.</p