Explore the vast range of titles available.

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.

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.

Cellular senescence is a potent anti-cancer mechanism controlled by tumor suppressor genes, particularly p53 and pRb, which is characterized by the irreversible loss of proliferation. Senescence induced by DNA damage, oncogenic stimulation, or excessive mitogenic input, serves as a barrier that counteracts cancer progression. Emerging evidence in cellular and in in vivo models revealed the involvement of additional signaling players in senescence, including PML, CK2, Bcl-2, PI3K effectors such as Rheb, Rho small GTPases, and cytokines. Recent studies have also implicated protein kinase C (PKC) isozymes as modulators of senescence phenotypes and showed that phorbol esters, widely used PKC activators, can induce senescence in a number of cancer cells. These novel findings suggest a complex array of cross-talks between senescence pathways and may have significant implications in cancer therapy.

The associations between the neurokinin-1 receptor (NK-1R), substance P (SP), and HIV-1 were investigated in neurosphere-derived cultures of microglial-depleted human fetal brain cells (HFBC). Full-length NK-1R was identified in HFBC cultures. SP treatment of the HFBC increased intracellular calcium mobilization and decreased electrical impedance, both of which were blocked by the NK-1R antagonist aprepitant. SP treatment of HIV-1-infected HFBC upregulated HIV-1 expression. These data show that human neural cells grown from neurospheres express functional full length NK-1R that is responsive to SP, and that SP enhanced HIV-1 infection in HBFC.

Nucleotides released during vascular injury from activated platelets and broken cells 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 the Gi/o inhibitor pertussis toxin (PTX) revealed that human neutrophils express only functional P2Y2 receptors. It was determined that the ability of nucleotides to cause elastase release from human neutrophils requires the presence of exogenous soluble fibrinogen. Neutrophils treated with phenidone and MK-886, inhibitors of 5-lipoxygenase pathway, showed that nucleotide-induced degranulation does not depend on generated arachidonic acid metabolites. Nucleotides caused activation of p38 MAPK and ERK 1/2 in human neutrophils. In addition, the p38 and ERK 1/2 MAPK inhibitors, SB203580 and U0126, inhibited nucleotide-induced elastase release. Therefore fibrinogen is required for nucleotide-induced primary granule release from human neutrophils through the P2Y2 receptor without a role for arachidonic acid metabolites. Both ERK 1/2 and p38 MAPK play an important role in nucleotide-induced primary granule release from human neutrophils. G protein coupling to the P2Y2 receptors and several downstream signaling events were investigated. Treatment of neutrophils with pertussis toxin (PTX) caused only a partial inhibition of Ca2+ mobilization indicating that unlike chemokines, such as fMLP, nucleotides lead to Ca 2+ mobilization through a Gi/o-independent pathway. This study determined that P2Y2 activation caused Ca2+ mobilization through activation of the PLC pathway for both the PTX-sensitive and the PTX-insensitive mechanism. The PLC inhibitor U73122 completely inhibited Ca2+ mobilization in both nucleotide- and fMLP-stimulated neutrophils. The phosphorylation of ERK was inhibited by PTX or U73122. The Ca 2+ 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. In conclusion, the P2Y2 receptor is able to cause Ca2+ mobilization through a PTX-insensitive but PLC-dependent pathway. ERK phosphorylation is mediated by a Gi/o-PLC pathway, that is independent of intracellular Ca2+-mobilization.