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

Integration of P2Y receptor-activated signal transduction pathways in G protein-dependent signalling networks

The role of nucleotides in intracellular energy provision and nucleic acid synthesis has been known for a long time. In the past decade, evidence has been presented that, in addition to these functions, nucleotides are also autocrine and paracrine messenger molecules that initiate and regulate a large number of biological processes. The actions of extracellular nucleotides are mediated by ionotropic P2X and metabotropic P2Y receptors, while hydrolysis by ecto-enzymes modulates the initial signal. An increasing number of studies have been performed to obtain information on the signal transduction pathways activated by nucleotide receptors. The development of specific and stable purinergic receptor agonists and antagonists with therapeutical potential largely contributed to the identification of receptors responsible for nucleotide-activated pathways. This article reviews the signal transduction pathways activated by P2Y receptors, the involved second messenger systems, GTPases and protein kinases, as well as recent findings concerning P2Y receptor signalling in C6 glioma cells. Besides vertical signal transduction, lateral cross-talks with pathways activated by other G protein-coupled receptors and growth factor receptors are discussed

Nucleotides potentiate the increase of free cytosolic calcium triggered by fMLP or IL-8 in a dose-dependent manner

<p><b>Copyright information:</b></p><p>Taken from "The priming effect of extracellular UTP on human neutrophils: Role of calcium released from thapsigargin-sensitive intracellular stores"</p><p></p><p>Purinergic Signalling 2005;1(4):359-368.</p><p>Published online Jan 2005</p><p>PMCID:PMC2096557.</p><p></p> (A) and (B) Human neutrophils were isolated and intracellular calcium increase was measured as described under Materials and methods. The effect of fMLP (10 nM) or IL-8 (10 nM) was measured when administered 2 min after the addition of different concentrations of UTP (circles) or ATP (squares). Averages ± SEM of peak intracellular calcium concentrations measured in 4–5 experiments are shown. (C) Representative tracings obtained in neutrophils stimulated with chemoattractant alone (10 nM fMLP or 10 nM IL-8) or with nucleotide (10 µM UTP or 10 µM ATP) followed by chemoattractant. The reagents were added to cell suspensions where indicated on each individual tracing

UTP potentiates fMLP- or IL-8-induced ROS production in a time-dependent manner

<p><b>Copyright information:</b></p><p>Taken from "The priming effect of extracellular UTP on human neutrophils: Role of calcium released from thapsigargin-sensitive intracellular stores"</p><p></p><p>Purinergic Signalling 2005;1(4):359-368.</p><p>Published online Jan 2005</p><p>PMCID:PMC2096557.</p><p></p> Human neutrophils were isolated and ROS production was measured as described under Materials and methods. (A) and (B) The effect of fMLP (10 nM) or IL-8 (10 nM) was measured when administered alone (empty bars) or simultaneously with 10 µM UTP (closed bars). (C) and (D) The effect of fMLP or IL-8 was measured when added at different intervals after the addition of UTP. Averages ± SEM of peak luminescence signal measured in 4–6 experiments are shown. The luminescence peak measured upon the addition of a final concentration of 200 µM of HO to medium containing isoluminol (5 µM) and horseradish peroxidase (1 U/ml) corresponds to 100 U on the -axes. (* < 0.05)

The effect of thapsigargin on the potentiating effect of UTP on fMLP-induced ROS production

<p><b>Copyright information:</b></p><p>Taken from "The priming effect of extracellular UTP on human neutrophils: Role of calcium released from thapsigargin-sensitive intracellular stores"</p><p></p><p>Purinergic Signalling 2005;1(4):359-368.</p><p>Published online Jan 2005</p><p>PMCID:PMC2096557.</p><p></p> Isolated neutrophils were incubated for 20 min with solvent (tracings in (A)) or 100 nM thapsigargin ( tracing in (B)). UTP (10 µM) and fMLP (10 nM) were added as indicated in each panel. Vertical bar corresponds to the luminescence peak measured upon the addition of a final concentration of 200 µM of HO to medium containing isoluminol (5 µM) and horseradish peroxidase (1 U/ml). Tracings are representative for data shown in Figure . (C) Thapsigargin (100 nM) was added to fura-2 loaded neutrophils resuspended in calcium-free medium. Intracellular calcium concentration was recorded as described under Materials and methods

UTP and ATP potentiate fMLP- or IL-8-induced ROS production in a dose-dependent manner

<p><b>Copyright information:</b></p><p>Taken from "The priming effect of extracellular UTP on human neutrophils: Role of calcium released from thapsigargin-sensitive intracellular stores"</p><p></p><p>Purinergic Signalling 2005;1(4):359-368.</p><p>Published online Jan 2005</p><p>PMCID:PMC2096557.</p><p></p> Human neutrophils were isolated and ROS production was measured as described under Materials and methods. The effect of 10 nM fMLP (A) or 10 nM IL-8 (B) was measured when administered alone or 1 min after the addition of increasing concentrations of UTP (circles) or ATP (squares). Averages ± SEM of peak luminescence signal measured in 3–5 experiments are shown. The luminescence peak measured upon the addition of a final concentration of 200 µM of HO to medium containing isoluminol (5 µM) and horseradish peroxidase (1 U/ml) corresponds to 100 U on the -axes. (* < 0.05 when compared to the values measured in the absence of nucleotides.