Intracellular Calcium Mobilization (intracellular + calcium_mobilization)

Distribution by Scientific Domains

Selected Abstracts

Vaccinia virus impairs directional migration and chemokine receptor switch of human dendritic cells


Abstract A crucial event for the induction of an anti-viral immune response is the coordinated, phenotype-dependent migration of dendritic cells (DC) to sites of infection and secondary lymphoid organs. Here we show that the vaccinia virus (VV) strains Western Reserve (WR) and modified virus Ankara (MVA) inhibit directional migration of mature DC toward the lymphoid chemokines CCL19 and CXCL12 without affecting surface expression of the respective chemokine receptors or impairing undirected cellular locomotion. Instead, infection with VV results in a deficiency of extracellular signal-regulated kinase-1 and a disturbance of intracellular calcium mobilization, indicating a viral interference with signaling events downstream of the surface chemokine receptors. In immature DC, apart from inhibiting chemokine-induced migration of infected DC, infection with both VV strains increases expression of the inflammatory chemokine receptors CCR1 and CXCR1 on non-infected bystander DC, which depends on the activity of IFN-,. Although functional, these chemokine receptors are resistant to lipopolysaccharide-induced down-regulation. In addition, VV-infected and non-infected bystander DC fail to up-regulate the lymphoid chemokine receptor CCR7 upon activation, together pointing to a disability to undergo the chemokine receptor switch. This study shows that VV targets directional migration of professional antigen-presenting cells at multiple functional levels, revealing a potent viral strategy of immune escape. See accompanying commentary: [source]

Characterization of a novel NCAM ligand with a stimulatory effect on neurite outgrowth identified by screening a combinatorial peptide library

Lars C. B. R°nn
Abstract The neural cell adhesion molecule, NCAM, plays a key role in neural development and plasticity mediating cell adhesion and signal transduction. By screening a combinatorial library of synthetic peptides with NCAM purified from postnatal day 10 rat brains, we identified a nonapeptide, termed NCAM binding peptide 10 (NBP10) and showed by nuclear magnetic resonance analysis that it bound the NCAM IgI module of NCAM. NBP10 modulated cell aggregation as well as neurite outgrowth induced specifically by homophilic NCAM binding. Moreover, both monomeric and multimeric forms of NBP10 stimulated neurite outgrowth from primary hippocampal neurons. The neurite outgrowth response to NBP10 was inhibited by a number of compounds previously shown to inhibit neurite outgrowth induced by homophilic NCAM binding, including voltage-dependent calcium channel antagonists, suggesting that NBP10 induced neurite outgrowth by activating a signal transduction pathway similar to that activated by NCAM itself. Moreover, an inhibitor of intracellular calcium mobilization, TMB-8, prevented NBP10-induced neurite outgrowth suggesting that NCAM-dependent neurite outgrowth also requires mobilization of calcium from intracellular calcium stores in addition to calcium influx from extracellular sources. By single-cell calcium imaging we further demonstrated that NBP10 was capable of inducing an increase in intracellular calcium in PC12E2 cells. Thus, the NBP10 peptide is a new tool for the study of molecular mechanisms underlying NCAM-dependent signal transduction and neurite outgrowth, and could prove to be a useful modulator of regenerative processes in the peripheral and central nervous system. [source]

15-Deoxy ,12,14 -prostaglandin J2 suppresses transcription by promoter 3 of the human thromboxane A2 receptor gene through peroxisome proliferator-activated receptor , in human erythroleukemia cells

FEBS JOURNAL, Issue 18 2005
Adrian T. Coyle
In humans, thromboxane (TX) A2 signals through two receptor isoforms, thromboxane receptor (TP), and TP,, which are transcriptionally regulated by distinct promoters, Prm1 and Prm3, respectively, within the single TP gene. The aim of the current study was to investigate the ability of the endogenous peroxisome proliferator-activated receptor (PPAR), ligand 15-deoxy-,12,14 -prostaglandin J2 (15d-PGJ2) to regulate expression of the human TP gene and to ascertain its potential effects on the individual TP, and TP, isoforms. 15d-PGJ2 suppressed Prm3 transcriptional activity and TP, mRNA expression in the platelet progenitor megakaryocytic human erythroleukemia (HEL) 92.1.7 cell line but had no effect on Prm1 or Prm2 activity or on TP, mRNA expression. 15d-PGJ2 also resulted in reductions in the overall level of TP protein expression and TP-mediated intracellular calcium mobilization in HEL cells. 15d-PGJ2 suppression of Prm3 transcriptional activity and TP, mRNA expression was found to occur through a novel mechanism involving direct binding of PPAR,,retinoic acid X receptor (RXR) heterodimers to a PPAR, response element (PPRE) composed of two imperfect hexameric direct repeat (DR) sequences centred at ,159 and ,148, respectively, spaced by five nucleotides (DR5). These data provide direct evidence for the role of PPAR, in the regulation of human TP gene expression within the vasculature and point to further critical differences in the modes of transcriptional regulation of TP, and TP, in humans. Moreover, these data highlight a further link between enhanced risk of cardiovascular disease in diabetes mellitus associated with increased synthesis and action of thromboxane A2 (TXA2). [source]

Gq/11-induced intracellular calcium mobilization mediates Per2 acute induction in Rat-1 fibroblasts

GENES TO CELLS, Issue 9 2006
Naoyuki Takashima
Phase resetting is one of the essential properties of circadian clocks that is required for the adjustment to a particular environment and the induction of Per1 and Per2 clock genes is believed to be a primary molecular event during this process. Although the intracellular signal transduction pathway underlying Per1 gene activation has been well characterized, the mechanisms that control Per2 up-regulation have not yet been elucidated. In our present study, we demonstrate that Gq/11 coupled receptors mediate serum-induced immediate rat Per2 (rPer2) transactivation in Rat-1 fibroblasts via intracellular Ca2+ mobilization. Stimulation of these cells with a high concentration of serum was found to rapidly increase the intracellular Ca2+ levels and strongly up-regulated rPer2 gene. rPer2 induction by serum stimulation was abrogated by intracellular Ca2+ chelation and depletion of intracellular Ca2+ store, which suggests that the calcium mobilization is necessary for the up-regulation of rPer2 gene. In addition, suppression of Gq/11 function was observed to inhibit both Ca2+ mobilization and rPer2 induction. Further, we demonstrated that endothelin-induced acute rPer2 transactivation via Gq/11-coupled endothelin receptors is also suppressed by a Gq/11 specific inhibitor. These findings together suggest that serum and endothelin utilize a common Gq/11-PLC mediated pathway for the transactivation of rPer2, which involves the mobilization of calcium from the intracellular calcium store. [source]

Activated ,2 macroglobulin induces matrix metalloproteinase 9 expression by low-density lipoprotein receptor-related protein 1 through MAPK-ERK1/2 and NF-,B activation in macrophage-derived cell lines

Leandro C. Cßceres
Abstract Macrophages under certain stimuli induce matrix metalloproteinase 9 (MMP-9) expression and protein secretion through the activation of MAPK-ERK and NF-,B signaling pathways. Previously, we demonstrated that activated ,2 -macroglulin (,2M*) through the interaction with its receptor low-density lipoprotein receptor-related protein 1 (LRP1) induces macrophage proliferation mediated by the activation of MAPK-ERK1/2. In the present work, we examined whether ,2M*/LRP1interaction could induce the MMP-9 production in J774 and Raw264.7 macrophage-derived cell lines. It was shown that ,2M* promoted MMP-9 expression and protein secretion by LRP1 in both macrophage-derived cell lines, which was mediated by the activation of MAPK-ERK1/2 and NF-,B. Both intracellular signaling pathways activated by ,2M* were effectively blocked by calphostin-C, suggesting involvement of PKC. In addition, we demonstrate that ,2M* produced extracellular calcium influx via LRP1. However, when the intracellular calcium mobilization was inhibited by BAPTA-AM, the ,2M*-induced MAPK-ER1/2 activation was fully blocked in both macrophage cell lines. Finally, using specific pharmacological inhibitors for PKC, Mek1, and NF-,B, it was shown that the ,2M*-induced MMP-9 protein secretion was inhibited, indicating that the MMP production promoted by the ,2M*/LRP1 interaction required the activation of both signaling pathways. These findings may prove useful in the understanding of the macrophage LRP1 role in the vascular wall during atherogenic plaque progression. J. Cell. Biochem. 111: 607,617, 2010. ę 2010 Wiley-Liss, Inc. [source]

Role of Protein Kinases in the Prolactin-Induced Intracellular Calcium Rise in Chinese Hamster Ovary Cells Expressing the Prolactin Receptor

B. Sorin
Abstract There is still only limited understanding of the early steps of prolactin signal transduction in target cells. It has been shown that prolactin actions are associated with cell protein phosphorylation, Ca2+ increases, and so on. However, the link between the activation of kinases and calcium influx or intracellular Ca2+ mobilization has not yet been clearly established. Chinese hamster ovary (CHO) cells, stably transfected with the long form of rabbit mammary gland prolactin receptor (PRL-R) cDNA were used for PRL-R signal transduction studies. Spectrofluorimetric techniques were used to measure intracellular calcium ([Ca2+]i) in cell populations with Indo1 as a calcium fluorescent probe. We demonstrate that, although protein kinase C activation (PMA or DiC8) caused a calcium influx in CHO cells, prolactin-induced PKC activation was not responsible for the early effect of prolactin on [Ca2+]i. Activation of protein kinase A (PKA) or protein kinase G did not modify [Ca2+]i and inhibition of PKA pathway did not affect the prolactin response. In the same way, phosphatidylinositol-3 kinaseinhibition had no effect on the prolactin-induced Ca2+ increase. On the other hand, tyrosine kinase inhibitors (herbimycin A, lavendustin A, and genistein) completely blocked the effect of prolactin on [Ca2+]i (influx and release). W7, a calmodulin-antagonist, and a specific inhibitor of calmodulin kinases (KN-62), only blocked prolactin-induced Ca2+ influx but had no significant effect on Ca2+ release. Using pharmacological agents, we present new data concerning the involvement of protein phosphorylations in the early effects of prolactin on ionic channels in CHO cells expressing the long form of PRL-R. Our results suggest that, at least in the very early steps of prolactin signal transduction, serine-threonine phosphorylation does not participate in the prolactin-induced calcium increase. On the other hand, tyrosine phosphorylation is a crucial, very early step, since it controls K+ channel activation, calcium influx, and intracellular calcium mobilization. Calmodulin acts later, since its inhibition only blocks the prolactin-induced Ca2+ influx. [source]

Ethanol Inhibits Muscarinic Receptor-Induced Axonal Growth in Rat Hippocampal Neurons

ALCOHOLISM, Issue 11 2009
Kathryn L. VanDeMark
Background:, In utero alcohol exposure can lead to fetal alcohol spectrum (FAS) disorders characterized by cognitive and behavioral deficits. In vivo and in vitro studies have shown that ethanol alters neuronal development. One mechanism through which ethanol has been shown to exert its effects is the perturbation of activated signaling cascades. The cholinergic agonist carbachol has been shown to induce axonal outgrowth through intracellular calcium mobilization, protein kinase C (PKC) activation, and ERK1/2 phosphorylation. This study investigated the effect of ethanol on the differentiation of rat hippocampal pyramidal neurons induced by carbachol as a possible mechanism involved in the developmental neurotoxicity of ethanol. Methods:, Prenatal rat hippocampal pyramidal neurons were treated with ethanol (50 to 75 mM) in the presence or absence of carbachol for 24 hours. Neurite outgrowth was assessed spectrophotometrically; axonal length was measured in neurons fixed and immunolabeled with the neuron-specific ,III tubulin antibody; cytotoxicity was analyzed using the thiazolyl blue tetrazolium bromide assay. The effect of ethanol on carbachol-stimulated intracellular calcium mobilization was assessed utilizing the fluorescent calcium probe, Fluo-3AM. The PepTag« assay for nonradioactive detection of PKC from Promega was used to measure PKC activity, and ERK1/2 activation was determined by densitometric analysis of Western blots probed for phospo-ERK1/2. Results:, Ethanol treatment (50 to 75 mM) caused an inhibition of carbachol-induced axonal growth, without affecting neuronal viability. Neuron treatment for 15 minutes with ethanol did not inhibit the carbachol-stimulated rise in intracellular calcium, while inhibiting PKC activity at the highest tested concentration and ERK1/2 phosphorylation at both the concentrations used in this study. On the other hand, neuron treatment for 24 hours with ethanol significantly inhibited carbachol-induced increase in intracellular calcium. Conclusions:, Ethanol inhibited carbachol-induced neurite outgrowth by inhibiting PKC and ERK1/2 activation. These effects may be, in part, responsible for some of the cognitive deficits associated with in utero alcohol exposure. [source]

Alcohol Suppresses IL-2,Induced CC Chemokine Production by Natural Killer Cells

ALCOHOLISM, Issue 9 2005
Ting Zhang
Background: Natural killer (NK) cells are a critical component of the host innate immune system. We investigated whether alcohol impairs NK cell function, particularly production of CC chemokines induced by interleukin (IL)-2, the natural ligands for CCR5 receptor. Methods: Primary NK cells and NK cell line (YTS) were cultured with or without alcohol (10 to 80 mM) for three hours. The culture supernatants were then harvested and used to treat human peripheral blood monocyte-derived macrophages and a HeLa cell line, which expresses CD4, CCR5, and CXCR4 receptors (MAGI cells). CC chemokine expression by YTS and primary NK cells treated with or without alcohol was analyzed with the real-time RT-PCR and ELISA. Ca2+i and Western blot assays were used to determine calcium-mediated intracellular signaling pathway and NF-,B p65 expression. HIV strains (Bal and UG024) were used to infect macrophages and MAGI cells. In addition, ADA (macrophage-tropic strain) and murine leukemia virus (MLV) envelope-pseudotyped HIV infection was carried out in macrophages. HIV infectivity was determined by HIV reverse transcriptase (RT) and ,-galactosidase activity assays. Results: Alcohol inhibited IL-2,induced CC chemokine (CCL3 and CCL4) expression by NK cells. Functional tests demonstrated that this reduced expression of CC chemokines was associated with diminished anti-HIV ability of NK cells. Alcohol also reduced the ability of NK cells to response to CCL3-mediated chemotaxis. Alcohol inhibited IL-2,induced NF-,B p65 protein expression and calcium mobilization by NK cells. Conclusions: Alcohol, through the inhibition of IL-2,induced NF-,B p65 protein expression and intracellular calcium mobilization, suppressed NK cell production of CC chemokines. This suppression of CC chemokine production was associated with diminished anti-HIV activity of NK cells. Thus, by inhibiting NK cell,mediated innate immunity against HIV, alcohol consumption may have a cofactor role in the immunopathogenesis of HIV disease. [source]

Anti-thrombotic effect of milrinone is caused by inhibition of calcium release from the dense tubular system in human platelets,

N. Hiramatsu
Aim: Milrinone, a phosphodiesterase III inhibitor, exerts positive inotropic effects which induce an increase in the intracellular calcium concentration by raising the cyclic adenosine monophosphate level in cardiac muscle. Milrinone was also reported to inhibit platelet aggregation, however, its mechanism remains unknown. Therefore, we investigated the effects of milrinone on intracellular calcium mobilization when platelets were activated. Methods: Washed platelets, obtained from six healthy volunteers, were preincubated with milrinone (0.9 ÁM) for 1 min and then exposed to 0.015 iÁ ml,1 thrombin for 5 min. The effect of milrinone on changes in the intracellular calcium level using a fluorescent dye, fura-2, was also observed. Calcium mobilizations via plasma membrane calcium channels and the dense tubular system were assessed differentially. Results: Milrinone (0.9 ÁM) significantly suppressed the aggregation ratios at 5 min compared with those in controls (86▒5%) to 75▒8%. The increase in the intracellular calcium concentration was also significantly suppressed (controls, 915▒293 nM vs. 405▒240 nM) when stimulated by thrombin. Milrinone also significantly inhibited the release of calcium from the dense tubular system (controls, 284▒111 nM vs. 158▒51 nM). Calcium influx through the plasma membrane was suppressed by milrinone 2.4 ÁM. Conclusion: Milrinone (0.9 ÁM) inhibited thrombin-induced platelet aggregation. This inhibitory effect was mainly mediated by suppressing calcium release from the dense tubular system. [source]

Protease-activated receptor-4 (PAR4): a role as inhibitor of visceral pain and hypersensitivity

C. AugÚ
Abstract, Protease-activated receptor-4 (PAR4) belongs to the family of receptors activated by the proteolytic cleavage of their extracellular N-terminal domain and the subsequent binding of the newly released N-terminus. While largely expressed in the colon, the role of PAR4 in gut functions has not been defined. We have investigated the effects of PAR4 agonist on colonic sensations and sensory neuron signalling, and its role in visceral pain. We observed that a single administration of the PAR4 agonist peptide (AYPGKF-NH2), but not the control peptide (YAPGKF-NH2) into the colon lumen of mice significantly reduced the visceromotor response to colorectal distension at different pressures of distension. Further, intracolonic administration of the PAR4 agonist, but not the control peptide, was able to significantly inhibit PAR2 agonist- and transcient receptor potential vanilloid-4 (TRPV4) agonist-induced allodynia and hyperalgesia in response to colorectal distension. Protease-activated receptor-4 was detected in sensory neurons projecting from the colon, and isolated from the dorsal root ganglia, where it co-expressed with PAR2 and TRPV4. In total sensory neurons, PAR4 agonist exposure inhibited free intracellular calcium mobilization induced by the pro-nociceptive agonists of PAR2 and TRPV4. Finally, PAR4 -deficient mice experienced increased pain behaviour in response to intracolonic administration of mustard oil, compared with wild-type littermates. These results show that PAR4 agonists modulate colonic nociceptive response, inhibit colonic hypersensitivity and primary afferent responses to pro-nociceptive mediators. Endogenous activation of PAR4 also plays a major role in controlling visceral pain. These results identify PAR4 as a previously unknown modulator of visceral nociception. [source]