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Ca2+ Mobilization (ca2+ + mobilization)
Kinds of Ca2+ Mobilization Selected AbstractsRoles of the actin-binding proteins in intracellular Ca2+ signallingACTA PHYSIOLOGICA, Issue 1 2009J. T. Chun Abstract Starfish oocytes undergo massive intracellular Ca2+ signalling during meiotic maturation and fertilization. Although the igniting stimulus of Ca2+ mobilization may differ in different cell contexts, its final leverage is usually the Ca2+ -releasing second messengers such as InsP3, cADPr and NAADP. The general scheme of intracellular Ca2+ release is that the corresponding receptors for these molecules serve as ion channels to release free Ca2+ from its internal stores such as the lumen of the endoplasmic reticulum. However, a growing body of evidence has suggested that intracellular Ca2+ release can be strongly modulated by the actin cytoskeleton. Although it is known that Ca2+ contributes to remodelling of the actin cytoskeleton, whether the actin cytoskeleton modulates Ca2+ signalling in return has not been much explored. An emerging candidate to answer to this reciprocal causality of Ca2+ and the actin cytoskeleton may be actin-binding proteins. In this review, we discuss how the actin cytoskeleton may fit into the known mechanisms of intracellular Ca2+ release, and propose two models to explain the experimental data. [source] Neuroprotective signal transduction in model motor neurons exposed to thrombin: G-protein modulation effects on neurite outgrowth, Ca2+ mobilization, and apoptosis ,DEVELOPMENTAL NEUROBIOLOGY, Issue 2 2001Irina V. Smirnova Abstract Thrombin, the ultimate protease in the blood coagulation cascade, mediates its known cellular effects by unique proteolytic activation of G-protein-coupled protease-activated receptors (PARs), such as PAR1, PAR3, and PAR4, and a "tethered ligand" mechanism. PAR1 is variably expressed in subpopulations of neurons and largely determines thrombin's effects on morphology, calcium mobilization, and caspase-mediated apoptosis. In spinal cord motoneurons, PAR1 expression correlates with transient thrombin-mediated [Ca2+]i flux, receptor cleavage, and elevation of rest [Ca2+]i activating intracellular proteases. At nanomolar concentrations, thrombin retracts neurites via PAR1 activation of the monomeric, 21 kDa Ras G-protein RhoA, which is also involved in neuroprotection at lower thrombin concentrations. Such results suggest potential downstream targets for thrombin's injurious effects. Consequently, we employed several G-protein-specific modulators prior to thrombin exposure in an attempt to uncouple both heterotrimeric and monomeric G-proteins from motoneuronal PAR1. Cholera toxin, stimulating Gs, and lovastatin, which blocks isoprenylation of Rho, reduced thrombin-induced calcium mobilization. In contrast, pertussis toxin and mastoparan, inhibiting or stimulating Go/Gi, were found to exacerbate thrombin action. Effects on neuronal rounding and apoptosis were also detected, suggesting therapeutic utility may result from interference with downstream components of thrombin signaling pathways in human motor neuron disorders, and possibly other neurodegenerative diseases. Published 2001 John Wiley & Sons, Inc. J Neurobiol 48: 87,100, 2001 [source] Two distinct P2Y receptors are involved in purine- and pyrimidine-evoked Ca2+ elevation in mammalian brain astrocytic culturesDRUG DEVELOPMENT RESEARCH, Issue 1-2 2001Chiara Bolego Abstract ATP and 2-methyl-thio-ATP (2-Me-SATP) increase cytosolic calcium concentrations ([Ca2+]i) in rat striatal astrocytes (Centemeri et al. [1997] Br J Pharmacol 121:1700,1706). The aim of the present study was to: (1) characterize pyrimidine-induced [Ca2+]i increases in the same experimental system, and (2) try to identify the multiple P2Y receptor subtypes mediating Ca2+ mobilization. UDP and UTP triggered a concentration-dependent [Ca2+]i elevation (EC50s = 0.58 ,M ± 0.4 and 31 ,M ± 6, respectively). Pyrimidine-evoked [Ca2+]i elevation was solely due to mobilization from intracellular stores, because: (1) removing calcium from extracellular medium or (2) blocking its influx with Ni2+ did not modify UTP responses; (3) the store-depleting agent thapsigargin completely abolished UTP-evoked [Ca2+]i increments. Guanosine-5,-O-(2-thiodiphosphate) partially inhibited the UTP response, whereas pertussis toxin (PTx) had no effect. The phospholipase C inhibitor U-73122 significantly reduced the UTP-evoked [Ca2+]i rise. Computer-assisted analysis indicated that the UTP and UDP responses are mediated by a single receptor, while ATP and 2-Me-SATP interact with two distinct receptors. The selective P2Y1 receptor antagonist MRS2179 abolished the ATP higher potency component. Sequential challenges with the same nucleotides resulted in almost complete homologous desensitization. Pre-exposure to UTP lowered the subsequent responses to either ATP or 2-Me-SATP. Maximally active concentrations of UTP and ATP were not additive. In conclusion, [Ca2+]i elevation in astrocytes by purines and pyrimidines is mediated by two distinct P2Y receptors, likely the P2Y1 and P2Y6 subtypes. Drug Dev. Res. 52:122,132, 2001. © 2001 Wiley-Liss, Inc. [source] Methyl mercury influences growth-related signaling in MCF-7 breast cancer cellsENVIRONMENTAL TOXICOLOGY, Issue 1 2005Olga A. Sukocheva Abstract Environmental contaminants have been shown to alter growth-regulating signaling pathways through molecular mechanisms that are mainly unclear. Here we report that within a narrow concentration range (0.5,1 ,M) methyl mercury (MeHg) significantly stimulated growth of MCF-7 cells, induced Ca2+ mobilization, and activated extracellular signal,regulated kinase ˝ (Erk1/2). MeHg modulated E2 -dependent stimulation of growth in a dose-dependent manner, although MeHg neither suppresses nor increases constitutive E2 metabolism. MeHg demonstrated weak estrogen receptor (ER),binding ability. However, long preincubation with antiestrogens LY156,758 and ICI164,384 decreased MeHg-induced foci formation, Ca2+ mobilization, and Erk1/2 activation, confirming involvement of ERs. The MeHg-induced increase in [Ca2+]i was observed to coincide with enhanced Erk1/2 phosphorylation. These data suggest that MeHg can significantly modulate the intracellular signaling environment in MCF-7 cells, resulting in a dose-dependent alteration of ER,mediated estrogenic capacity and therefore should be considered as a potential estrogen-disrupting compound. © 2005 Wiley Periodicals, Inc. Environ Toxicol 20: 32,44, 2005. [source] Constitutive activation of Bruton's tyrosine kinase induces the formation of autoreactive IgM plasma cellsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 9 2010Rogier Kersseboom Abstract B-cell receptor (BCR)-mediated signals provide the basis for B-cell differentiation in the BM and subsequently into follicular, marginal zone, or B-1 B-cell subsets. We have previously shown that B-cell-specific expression of the constitutive active E41K mutant of the BCR-associated molecule Bruton's tyrosine kinase (Btk) leads to an almost complete deletion of immature B cells in the BM. Here, we report that low-level expression of the E41K or E41K-Y223F Btk mutants was associated with reduced follicular B-cell numbers and significantly increased proportions of B-1 cells in the spleen. Crosses with 3-83,, and VH81X BCR Tg mice showed that constitutive active Btk expression did not change follicular, marginal zone, or B-1 B-cell fate choice, but resulted in selective expansion or survival of B-1 cells. Residual B cells were hyperresponsive and manifested sustained Ca2+ mobilization. They were spontaneously driven into germinal center-independent plasma cell differentiation, as evidenced by increased numbers of IgM+ plasma cells in spleen and BM and significantly elevated serum IgM. Because anti-nucleosome autoantibodies and glomerular IgM deposition were present, we conclude that constitutive Btk activation causes defective B-cell tolerance, emphasizing that Btk signals are essential for appropriate regulation of B-cell activation. [source] Brain-derived neurotrophic factor induces long-lasting Ca2+ -activated K+ currents in rat visual cortex neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2002Yoshito Mizoguchi Abstract Brain-derived neurotrophic factor (BDNF) increases postsynaptic intracellular Ca2+ and modulates synaptic transmission in various types of neurons. Ca2+ -activated K+ currents, opened mainly by intracellular Ca2+ elevation, contribute to hyperpolarization following action potentials and modulate synaptic transmission. We asked whether BDNF induces Ca2+ -activated K+ currents by postsynaptic elevation of intracellular Ca2+ in acutely dissociated visual cortex neurons of rats. Currents were analysed using the nystatin-perforated patch clamp technique and imaging of intracellular Ca2+ mobilization with fura-2. At a holding potential of ,50 mV, BDNF application (20 ng/mL) for 1,2 min induced an outward current (IBDNF-OUT; 80.0 ± 29.0 pA) lasting for more than 90 min without attenuation in every neuron tested. K252a (200 nm), an inhibitor of Trk receptor tyrosine kinase, and U73122 (3 ,m), a specific phospholipase C (PLC)-, inhibitor, suppressed IBDNF-OUT completely. IBDNF-OUT was both charybdotoxin- (600 nm) and apamin- (300 nm) sensitive, suggesting that this current was carried by Ca2+ -activated K+ channels. BAPTA-AM (150 ,m) gradually suppressed IBDNF-OUT. Fura-2 imaging revealed that a brief application of BDNF elicited a long-lasting elevation of intracellular Ca2+. These results show that BDNF induces long-lasting Ca2+ -activated K+ currents by sustained intracellular Ca2+ elevation in rat visual cortex neurons. While BDNF, likely acting through the Trk B receptor, was necessary for the induction of long-lasting Ca2+ -activated K+ currents via intracellular Ca2+ elevation, BDNF was not necessary for the maintenance of this current. [source] Calcium-sensing receptor mediates phenylalanine-induced cholecystokinin secretion in enteroendocrine STC-1 cellsFEBS JOURNAL, Issue 18 2008Tohru Hira Intraluminal l -phenylalanine (Phe) stimulates cholecystokinin (CCK) secretion in vivo and in vitro. However, the cellular mechanism by which CCK-producing enteroendocrine cells sense Phe is unknown. The calcium-sensing receptor (CaR) can sense amino acids, and is expressed in the gastrointestinal tract. In the present study, we examined whether CaR functions as a receptor for Phe in CCK-producing enteroendocrine cells. CCK secretion and intracellular Ca2+ concentration in response to Phe were measured in the murine CCK-producing enteroendocrine cell line STC-1 at various extracellular Ca2+ concentrations or after treatment with a CaR antagonist. At more than 20 mm, Phe induced dose-dependent CCK secretion and intracellular Ca2+ mobilization in STC-1 cells. In the presence of 3.0 mm extracellular Ca2+, 10 and 20 mm Phe induced significantly higher CCK secretion than under normal conditions (1.2 mm extracellular Ca2+). Intracellular Ca2+ mobilization, induced by 10 or 20 mm Phe, was also enhanced by increasing extracellular Ca2+ concentrations. In addition, intracellular Ca2+ mobilization induced by addition of extracellular Ca2+ was augmented by the presence of Phe. These results closely match the known CaR properties. Treatment with a specific CaR antagonist (NPS2143) completely inhibited Phe-induced CCK secretion and the latter phase of intracellular Ca2+ mobilization. CaR mRNA expression was demonstrated by RT-PCR in STC-1 cells, as well as in other mouse tissues including the kidney, thyroid, stomach and intestine. In conclusion, CaR functions as a receptor for Phe, stimulating CCK secretion in enteroendocrine STC-1 cells. [source] Gq/11-induced intracellular calcium mobilization mediates Per2 acute induction in Rat-1 fibroblastsGENES TO CELLS, Issue 9 2006Naoyuki 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] Nucleotide-induced Ca2+ signaling in sustentacular supporting cells of the olfactory epitheliumGLIA, Issue 15 2008Thomas Hassenklöver Abstract Extracellular purines and pyrimidines are important signaling molecules acting via purinergic cell-surface receptors in neurons, glia, and glia-like cells such as sustentacular supporting cells (SCs) of the olfactory epithelium (OE). Here, we thoroughly characterize ATP-induced responses in SCs of the OE using functional Ca2+ imaging. The initial ATP-induced increase of the intracellular Ca2+ concentration [Ca2+]i always occurred in the apical part of SCs and subsequently propagated toward the basal lamina, indicating the occurrence of purinergic receptors in the apical part of SCs. The mean propagation velocity of the Ca2+ signal within SCs was 17.10 ± 1.02 ,m/s. ATP evoked increases in [Ca2+]i in both the presence and absence of extracellular Ca2+. Depletion of the intracellular Ca2+ stores abolished the responses. This shows that the ATP-induced [Ca2+]i increases were in large part, if not entirely, due to the activation of G protein-coupled receptors followed by Ca2+ mobilization from intracellular stores, suggesting an involvement of P2Y receptors. The order of potency of the applied purinergic agonists was UTP > ATP > ATP,S (with all others being only weakly active or inactive). The ATP-induced [Ca2+]i increases could be reduced by the purinergic antagonists PPADS and RB2, but not by suramin. Our findings suggest that extracellular nucleotides in the OE activate SCs via P2Y2/P2Y4 -like receptors and initiate a characteristic intraepithelial Ca2+ wave. © 2008 Wiley-Liss, Inc. [source] Confocal imaging and tracking of the exocytotic routes for D -serine-mediated gliotransmissionGLIA, Issue 12 2008Magalie Martineau Abstract D -Serine is an astrocyte-derived regulator for N -methyl- D -aspartate receptors, but the intracellular routes of its trafficking are still largely unknown. Here, we combined confocal microscopy with colocalization quantification to track the astrocytic organelles that store D -serine. We report that D -serine colocalizes with the transfected eGFP-synaptobrevin/VAMP2 and eGFP-cellubrevin/VAMP3, two v-SNAREs of the regulated secretory pathway. No significant colocalization was found with markers of the endosomal sorting and recycling system: EEA1, eGFP-endobrevin/VAMP8, eGFP-TI-VAMP/VAMP7, LAMP1, and CD63. Blockade of vesicular budding with colchicine shows that secretory vesicles import D -serine downstream to the Golgi apparatus. Finally, treatment of astrocytes with the Ca2+ -ionophore A23187, glutamate agonists, or bradykinin trigger translocation of synaptobrevin/VAMP2 to the plasma membrane with a concomitant disappearance of D -serine from the regulated secretory pathway. Our results provide morphological evidence for a vesicular storage of D -serine in the regulated secretory pathway and the possible recruitment of these stores by Ca2+ mobilization to release D -serine. © 2008 Wiley-Liss, Inc. [source] Effects of prolactin on intracellular calcium concentration and cell proliferation in human glioma cellsGLIA, Issue 3 2002Thomas Ducret Abstract Prolactin (PRL) has several physiological effects on peripheral tissues and the brain. This hormone acts via its membrane receptor (PRL-R) to induce cell differentiation or proliferation. Using reverse transcription,polymerase chain reaction (RT-PCR) combined with Southern blot analysis, we detected PRL-R transcripts in a human glioma cell line (U87-MG) and in primary cultured human glioblastoma cells. These transcripts were deleted or not in their extracellular domains. We examined the effects of PRL on intracellular free Ca2+ concentration ([Ca2+]i) in these cells in order to improve our understanding of the PRL transduction mechanism, which is still poorly documented. [Ca2+]i was measured by microspectrofluorimetry using indo-1 as the Ca2+ fluorescent probe. Spatiotemporal aspects of PRL-induced Ca2+ signals were investigated using high-speed fluo-3 confocal imaging. We found that physiological concentrations (0.4,4 nM) of PRL-stimulated Ca2+ entry and intracellular Ca2+ mobilization via a tyrosine kinase,dependent mechanism. The two types of Ca2+ responses observed were distinguishable by their kinetics: one showing a slow (type I) and the other a fast (type II) increase in [Ca2+]i. The amplitude of PRL-induced Ca2+ increases may be sufficient to provoke several physiological responses, such as stimulating proliferation. Furthermore, PRL induced a dose-dependent increase in [3H]thymidine incorporation levels and in cellular growth and survival, detected by the MTT method. These data indicate that PRL induced mitogenesis of human glioma cells. GLIA 38:200,214, 2002. © 2002 Wiley-Liss, Inc. [source] Basic Fibroblast Growth Factor Stimulates Vascular Endothelial Growth Factor Release in Osteoblasts: Divergent Regulation by p42/p44 Mitogen-Activated Protein Kinase and p38 Mitogen-Activated Protein KinaseJOURNAL OF BONE AND MINERAL RESEARCH, Issue 12 2000Haruhiko Tokuda Abstract We previously showed that basic fibroblast growth factor (bFGF) activates p38 mitogen-activated protein (MAP) kinase via Ca2+ mobilization, resulting in interleukin-6 (IL-6) synthesis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of bFGF on the release of vascular endothelial growth factor (VEGF) in these cells. bFGF stimulated VEGF release dose dependently in the range between 10 and 100 ng/ml. SB203580, an inhibitor of p38 MAP kinase, markedly enhanced the bFGF-induced VEGF release. bFGF induced the phosphorylation of both p42/p44 MAP kinase and p38 MAP kinase. PD98059, an inhibitor of upstream kinase of p42/p44 MAP kinase, reduced the VEGF release. SB203580 enhanced the phosphorylation of p42/p44 MAP kinase induced by bFGF. The enhancement by SB203580 of the bFGF-stimulated VEGF release was suppressed by PD98059. The depletion of extracellular Ca2+ by [ethylenebis-(oxyethylenenitrilo)]tetracetic acid (EGTA) or 1,2-bis-(O -aminophinoxy)-ethane- N,N,N,N -tetracetic acid tetracetoxymethyl ester (BAPTA/AM), a chelator of intracellular Ca2+, suppressed the bFGF-induced VEGF release. A23187, a Ca ionophore, or thapsigargin, known to induce Ca2+ release from intracellular Ca2+ store, stimulated the release of VEGF by itself. A23187 induced the phosphorylation of p42/p44 MAP kinase and p38 MAP kinase. PD98059 suppressed the VEGF release induced by A23187. SB203580 had little effect on either A23187-induced VEGF release or the phosphorylation of p42/p44 MAP kinase by A23187. These results strongly suggest that bFGF stimulates VEGF release through p42/p44 MAP kinase in osteoblasts and that the VEGF release is negatively regulated by bFGF-activated p38 MAP kinase. [source] Role of LOX-1 in monocyte adhesion-triggered redox, Akt/eNOS and Ca2+ signaling pathways in endothelial cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2009Nobuo Sakamoto This study was conducted to examine the role of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in monocyte adhesion-induced redox-sensitive, Akt/eNOS and Ca2+ signaling pathways in endothelial cells (ECs). LOX-1 was blocked by an antibody-neutralizing LOX-1 TS92 or small interfering RNA. In cultured human aortic ECs, monocyte adhesion activated Rac1 and p47phox, and increased NADPH oxidase activity and reactive oxygen species (ROS) generation within 30,min and NF-,B phosphorylation within 1,h, resulting in redox-sensitive gene expression. Akt and eNOS phosphorylation was induced 15,min after adding monocytes and returned to control level after 30,min, whereas NO production was not altered by monocyte adhesion. Blockade of LOX-1 blunted the monocyte adhesion-triggered redox-sensitive signaling pathway and Akt/eNOS phosphorylation in ECs. Both endothelial intracellular Ca2+ mobilization and Ca2+ influx caused by monocyte attachment were markedly attenuated by pretreatment of ECs with TS92. This suggests that LOX-1 is involved in redox-sensitive, Akt/eNOS and Ca2+ signaling pathways in monocyte adhesion to ECs independent of oxidized low-density lipoprotein (ox-LDL). Furthermore, blockade of Ca2+ inhibited monocyte adhesion-triggered Rac1 and p47phox activation and ROS generation in ECs, whereas Ca2+ signaling was suppressed by blockade of NADPH oxidase and ROS generation. Finally, TS92 blocked the monocyte adhesion to ECs stimulated with or without tumor necrosis factor-, or ox-LDL. We provide evidence that LOX-1 plays a role in redox-sensitive, Akt/eNOS and Ca2+ signaling pathways in monocyte adhesion to ECs independent of the ox-LDL,LOX-1 axis. J. Cell. Physiol. 220: 706,715, 2009. © 2009 Wiley-Liss, Inc. [source] Sphingosine 1-phosphate induces cell contraction via calcium-independent/Rho-dependent pathways in undifferentiated skeletal muscle cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2004L. Formigli We have previously shown that sphingosine 1-phosphate (S1P) can induce intracellular Ca2+ mobilization and cell contraction in C2C12 myoblasts and that the two phenomena are temporally unrelated. Although Ca2+ -independent mechanisms of cell contraction have been the focus of numerous studies on Ca2+ sensitization of smooth muscle, comparatively less studies have focused on the role that these mechanisms play in the regulation of skeletal muscle contractility. Phosphorylation and activation of myosin by Rho-dependent kinase mediate most of Ca2+ -independent contractile responses. In the present study, we examined the potential role of Rho/Rho-kinase cascade activation in S1P-induced C2C12 cell contraction. First, we showed that depletion of Ca2+, by pre-treatment with BAPTA, did not affect S1P-induced myoblastic contractility, whereas it abolished S1P-induced Ca2+ transients. These results correlated with the absence of troponin C and with the immature cytoskeletal organization of these cells. Experimental evidence demonstrating the involvement of Rho pathway in S1P-stimulated myoblast contraction included: the activation/translocation of RhoA to the membrane in response to agonist-stimulation in cells depleted of Ca2+ and the inhibition of dynamic changes of the actin cytoskeleton in cells where Rho functions had been inhibited either by overexpression of RhoGDI, a physiological inhibitor of GDP dissociation from Rho proteins, or by pretreatment with Y-27632, a specific Rho kinase inhibitor. Contribution of protein kinase C in this cytoskeletal rearrangement was also evaluated. However, the pretreatment with Gö6976 or rottlerin, specific inhibitors of PKC, and PKC,, respectively, failed to inhibit the agonist-induced myoblastic contraction. Single particle tracking of G-actin fluorescent probe was performed to statistically evaluate actin cytoskeletal dynamics in response to S1P. Stimulation with S1P was also able to increase the phosphorylation level of myosin light chain II. In conclusion, our results strongly suggest that Ca2+ -independent/Rho-Rho kinase-dependent pathways may exert an important role in S1P-induced myoblastic cell contraction. J. Cell. Physiol. 198: 1,11, 2004© 2003 Wiley-Liss, Inc. [source] Recombinant human serotonin 5A receptors stably expressed in C6 glioma cells couple to multiple signal transduction pathwaysJOURNAL OF NEUROCHEMISTRY, Issue 2 2003Mami Noda Abstract Human serotonin 5A (5-HT5A) receptors were stably expressed in undifferentiated C6 glioma. In 5-HT5A receptors-expressing cells, accumulation of cAMP by forskolin was inhibited by 5-HT as reported previously. Pertussis toxin-sensitive inhibition of ADP-ribosyl cyclase was also observed, indicating a decrease of cyclic ADP ribose, a potential intracellular second messenger mediating ryanodine-sensitive Ca2+ mobilization. On the other hand, 5-HT-induced outward currents were observed using the patch-clamp technique in whole-cell configuration. The 5-HT-induced outward current was observed in 84% of the patched 5-HT5A receptor-expressing cells and was concentration-dependent. The 5-HT-induced current was inhibited when intracellular K+ was replaced with Cs+ but was not significantly inhibited by typical K+ channel blockers. The 5-HT-induced current was significantly attenuated by 1,2-bis(2-aminophenoxy)ethane- N,N,N,,N,-tetraacetic acid (BAPTA) in the patch pipette. Depleting intracellular Ca2+ stores by application of caffeine or thapsigargin also blocked the 5-HT-induced current. Blocking G protein, the inositol triphosphate (IP3) receptor, or pretreatment with pertussis toxin, all inhibited the 5-HT-induced current. IP3 showed a transient increase after application of 5-HT in 5-HT5A receptor-expressing cells. It was concluded that in addition to the inhibition of cAMP accumulation and ADP-ribosyl cyclase activity, 5-HT5A receptors regulate intracellular Ca2+ mobilization which is probably a result of the IP3-sensitive Ca2+ store. These multiple signal transduction systems may induce complex changes in the serotonergic system in brain function. [source] Role of Protein Kinases in the Prolactin-Induced Intracellular Calcium Rise in Chinese Hamster Ovary Cells Expressing the Prolactin ReceptorJOURNAL OF NEUROENDOCRINOLOGY, Issue 9 2000B. 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] Histamine-induced Ca2+ entry in human astrocytoma U373 MG cells: Evidence for involvement of store-operated channelsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 15 2008Margarita Barajas Abstract Glial and glia-derived cells express a variety of receptors for neurotransmitters and hormones, the majority of which evoke both Ca2+ release from intracellular stores and Ca2+ entry across the plasma membrane. We investigated the links between histamine H1 receptor activation, Ca2+ release from intracellular stores and Ca2+ influx in human astrocytoma U373 MG cells. Histamine, through a H1 receptor-mediated effect, evoked an increase in cytoplasmic free calcium concentration ([Ca2+]i) that occurred in two phases: an initial, transient, increase owing to Ca2+ mobilization from intracellular pools, and a second, sustained increase dependent on both Ca2+ influx and continuous receptor occupancy. The characteristics of histamine-induced increases in [Ca2+]i were similar to the capacitative entry evoked by emptying of the Ca2+ stores with thapsigargine, and different from that observed when Ca2+ influx was activated with OAG (1-oleoyl-2-acetyl- sn -glycerol), a diacylglycerol (DAG) analog. OAG application or increased endogenous DAG, resulting from DAG kinase inhibition, reduced the histamine-induced response. Furthermore, activation of the DAG target, protein kinase C (PKC), by TPA (12-O-tetradecanoyl 4,-phorbol 13,-acetate) resulted in inhibition of the histamine-induced Ca2+ response, an action prevented by PKC inhibitors. By using reverse transcriptase,polymerase chain reaction analysis, mRNAs for transient receptor potential channels (TRPCs) 1, 4, and 6 as well as for STIM1 (stromal-interacting molecule) and Orai1 were found to be expressed in the U373 MG cells, and confocal microscopy using specific antibodies revealed the presence of the corresponding proteins. Therefore, TRPCs may be candidate proteins forming store-operated channels in the U373 MG cell line. Further, our results confirm the involvement of PKC in the regulation of H1 receptor-induced responses and point out to the existence of a feedback mechanism acting via PKC to limit the increase in [Ca2+]i. © 2008 Wiley-Liss, Inc. [source] Ca2+ mobilization mediated by transient receptor potential canonical 3 is associated with thrombin-induced morphological changes in 1321N1 human astrocytoma cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 12 2008Kenji Nakao Abstract Activated astrocytes show various patterns of Ca2+ mobilization under pathological conditions. In the present study we revealed a novel function of astrocytic Ca2+ dynamics through investigation of thrombin-induced unique Ca2+ entry. Using 1321N1 human astrocytoma cells, which have been shown to be a good model for detecting morphological dynamics, we observed rapid retraction of bipolar protrusions that were reversibly evoked by 0.03,3 U/mL thrombin. Morphological changes were predominantly dependent on a specific thrombin receptor subtype, proteinase-activated receptor 1 (PAR-1). In parallel, Fura-2 imaging of intracellular Ca2+ concentration ([Ca2+]i) showed that thrombin induced heterogeneous Ca2+ responses with asynchronous repetitive peaks. These oscillations were found to be a result of repetitive Ca2+ release from intracellular stores, followed by refilling of Ca2+ from the extracellular region without a direct [Ca2+]i increase. Pharmacological manipulation with BAPTA-AM, cyclopiazonic acid, and 2-aminoethoxydiphenyl borate indicated that Ca2+ mobilization was involved in thrombin-induced morphological changes. We further addressed the role of Ca2+ entry using small interfering RNA (siRNA) for transient receptor potential canonical 3 (TRPC3). As a result, both thrombin-induced morphological changes and oscillatory Ca2+ responses were significantly attenuated in siRNA-transfected cells. Inhibition of TRPC3 with pyrazole-3 also provided support for the contribution of Ca2+ influx. Moreover, TRPC3-mediated Ca2+ dynamics regulated thrombin-induced phosphorylation of myosin light chain 2. These results suggest a novel function of astrocytic Ca2+ dynamics, including Ca2+ entry, in the pathophysiological effects of PAR-1-mediated astrocytic activation. TRPC3 forms a functional Ca2+ channel and might modulate astrocytic activation in response to brain hemorrhaging. © 2008 Wiley-Liss, Inc. [source] Structural properties of orexins for activation of their receptorsJOURNAL OF PEPTIDE SCIENCE, Issue 4 2006Manja Lang Abstract The closely related neuropeptides orexin A and orexin B mediate their actions, including the regulation of sleep and appetite, by the activation of the orexin 1 and 2 receptors. To elucidate the structural prerequisites for receptor activation and subtype selectivity, we performed multiple amino acid substitutions within the sequence of orexin A and human orexin B-(6-28)-peptide and analyzed their solution structures by CD spectroscopy and their activity at both receptors in Ca2+ mobilization assays. For orexin A, we showed that the basic amino acids within the segment of residues 6,14 were important for the activation of both receptors. Furthermore, we showed that the restriction via disulfide bonds is not required to maintain the active structure of orexin A. The kink region of h orexin B has been shown to be important for Ox2R selectivity, which is not mediated by the restriction of the turn structure. Additionally, we showed that no particular secondary structure is required for receptor subtype selectivity. Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd. [source] Areca nut extracts-activated secretion of leukotriene B4, and phosphorylation of p38 mitogen-activated protein kinase and elevated intracellular calcium concentrations in human polymorphonuclear leukocytesJOURNAL OF PERIODONTAL RESEARCH, Issue 5 2007S.-L. Hung Background and Objective:, Polymorphonuclear leukocytes are the major source of leukotriene B4, which is synthesized via the 5-lipoxygenase pathway. Activation of the 5-lipoxygenase pathway is regulated by intracellular calcium and the phosphorylation of p38 mitogen-activated protein kinase (MAPK). The impact of areca nut extracts on the biosynthesis of leukotriene B4 by human polymorphonuclear leukocytes was evaluated, and some of the possible mechanisms underlying the responses were examined. Material and Methods:, Polymorphonuclear leukocytes were treated with various concentrations of areca nut extracts. The concentrations of leukotriene B4 released into the supernatants were evaluated using enzyme immunoassay. The phosphorylation of p38 MAPK was monitored using immunoblotting, and the cytosolic calcium kinetics were assessed fluorometrically using Fura-2. Results:, Exposure of polymorphonuclear leukocytes to areca nut extracts led to a dose-dependent increase in the production of leukotriene B4, with levels peaking at 30 min and decreasing thereafter. Areca nut extracts enhanced the phosphorylation of p38 MAPK, an enzyme known to activate 5-lipoxygenase. Incubation with areca nut extracts also resulted in a rapid elevation of intracellular calcium concentrations in polymorphonuclear leukocytes. The induction of leukotriene B4 by areca nut extracts was suppressed with the p38 MAPK inhibitor, SB203580, or with the intracellular calcium chelator, BAPTA-AM. Conclusion:, The interaction of areca nut extracts with polymorphonuclear leukocytes activated the arachidonic acid metabolic cascade. Incubation of polymorphonuclear leukocytes with areca nut extracts resulted in the activation of intracellular events, such as phosphorylation of p38 MAPK and Ca2+ mobilization, involved in the release of pro-inflammatory lipid mediators. The results of this study emphasize the potential importance of polymorphonuclear leukocytes as a source of leukotriene B4, which may modulate the inflammatory response in areca chewers. [source] Prostaglandin F2, upregulates interleukin-6 production in human gingival fibroblastsJOURNAL OF PERIODONTAL RESEARCH, Issue 2 2001Kazuyuki Noguchi Prostaglandin F2,(PGF2,) is a bioactive lipid mediator which has been suggested to be involved in the pathogenesis of periodontal disease. However, the roles of PGF2, in periodontal lesions are poorly understood. In the present study, we investigated the effect of PGF2, on interleukin (IL)-6 production in human gingival fibroblasts (HGF). PGF2,stimulated IL-6 production in a time- and concentration-dependent fashion. IL-1, and tumor necrosis factor ,(TNF,), proinflammatory cytokines, induced IL-6 production in a time-dependent manner, and PGF2,synergistically enhanced IL-6 production induced by IL-1, and TNF,. IL-6 mRNA was expressed in PGF2, -stimulated HGF, and PGF2, increased IL-6 mRNA levels induced by IL-1, and TNF,. Fluprostenol, a selective FP receptor agonist, could mimic PGF2, -induced IL-6 production. Since FP receptors are coupled to elevation of intracellular calcium and activation of protein kinase C (PKC), the mechanism of IL-6 production by PGF2, was investigated using TMB-8, an inhibitor of Ca2+ mobilization from intracellular stores, and calphostin C, an inhibitor of PKC. TMB-8 significantly suppressed PGF2, -induced IL-6 production, whereas calphostin C showed a stimulatory effect on PGF2, -induced IL-6 production. From these data, we suggest that PGF2, upregulates IL-6 production through FP receptors in HGF, that PGF2, synergistically enhances IL-6 production in IL-1,- and TNF,-stimulated HGF, and that PGF2, -induced IL-6 production may be dependent on intracellular Ca2+ mobilization and be downregulated by PKC activation. PGF2, may be involved in the pathogenesis of periodontal disease by enhancing IL-6 levels in periodontal lesions. [source] Products of tryptophan catabolism induce Ca2+ release and modulate the cell cycle of Plasmodium falciparum malaria parasitesJOURNAL OF PINEAL RESEARCH, Issue 3 2005Flávio H. Beraldo Abstract:, Intraerythrocytic malaria parasites develop in a highly synchronous manner. We have previously shown that the host hormone melatonin regulates the circadian rhythm of the rodent malaria parasite, Plasmodium chabaudi, through a Ca2+ -based mechanism. Here we show that melatonin and other molecules derived from tryptophan, i.e. N -acetylserotonin, serotonin and tryptamine, also modulate the cell cycle of human malaria parasite P. falciparum by inducing an increase in cytosolic free Ca2+. This occurs independently of the extracellular Ca2+ concentration, indicating that these molecules induce Ca2+ mobilization from intracellular stores in the trophozoite. This in turn leads to an increase in the proportion of schizonts. The effects of the indolamines in increasing cytosolic free Ca2+ and modulating the parasite cell cycle are both abrogated by an antagonist of the melatonin receptor, luzindole, and by the phospholipase inhibitor, U73122. [source] von Willebrand factor activates endothelial nitric oxide synthase in blood platelets by a glycoprotein Ib-dependent mechanismJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 12 2006R. RIBA Summary.,Background: The molecular regulation of endothelial nitric oxide synthase (eNOS) in blood platelets and the signalling events induced by platelet-derived NO are poorly defined. In particular, the ability of von Willebrand factor (VWF) to stimulate cyclic guanosine monophosphate (cGMP) formation in platelets has produced conflicting data. Objectives: To determine the mechanisms leading to eNOS activation and clarify the downstream signaling pathways activated by platelet-derived NO in response to VWF. Methods: We used three independent markers of NO signaling, [3H] l -citrulline production, cGMP accrual and immunoblotting of vasodilator,stimulated phosphoprotein (VASP) to examine the NO signaling cascade in response to VWF. Results: VWF increased NO synthesis and bioavailability, as evidenced by increased [3H] l -citrulline production and cGMP accrual, respectively. VWF-induced eNOS activation was GPIb-IX-dependent and independent of integrin ,IIb,3. cGMP formation in response to VWF required Ca2+ mobilization, Src family kinases, phosphatidylinositol 3-kinase and phospholipase C, but not protein kinase C. This suggests that a cross-talk between the signaling mechanisms regulates platelet activation and NO synthesis. VWF-induced cGMP accrual was completely blocked by apyrase and indomethacin, demonstrating an essential role for platelet-derived ADP and thromboxane A2 (TxA2). Elevated cGMP levels led to increased VASP phosphorylation at serine239 that was both protein kinase G (PKG)- and protein kinase A (PKA)-dependent. Conclusions: We demonstrate that VWF activates eNOS through a specific Ca2+ -dependent GPIb receptor-signaling cascade that relies on the generation of platelet-derived ADP and TxA2. Furthermore, we provide the first evidence to suggest that platelet derived-NO/cGMP activates PKA in addition to PKG. [source] Mite serine protease activates protease-activated receptor-2 and induces cytokine release in human keratinocytesALLERGY, Issue 9 2009T. Kato Background:, House dust mites produce serine and cysteine proteases. Mite-derived proteases have been suggested to be involved in the pathogenesis of allergies; however, whether mite-derived serine protease activity can stimulate keratinocytes remains unknown. Methods:, We examined the activation of primary human keratinocytes by serine protease-rich extract of whole mite culture and compared with that by recombinant group 1 allergens (rDer f 1 and rDer p 1), which exclusively exhibit cysteine protease activity. Results:, Protease activity of whole mite culture extract (WCE), rDer f 1 and rDer p 1 induced the release of IL-8 and granulocyte-macrophage colony-stimulating factor. Protease activity of WCEs induced a significant upregulation of their mRNA expression but rDer f 1 had much less effect. Protease activity of the WCE stimulated intracellular Ca2+ mobilization but rDer f 1 and rDer p 1 did not. The mobilization induced by agonists for the human protease-activated receptor (PAR)-2, an agonist peptide or trypsin, was diminished by pre-incubation of keratinocytes with WCE. rDer f 1 inefficiently cleaved a synthetic N-terminal peptide of PAR-2 at different sites from trypsin, but the resultant peptides did not stimulate the release of interleukin-8. Conclusions:, The results suggest that mite-derived serine protease activity may contribute to the pathogenesis of atopic dermatitis by activating keratinocytes via PAR-2 activation but cysteine protease activity of Der f 1 and Der p 1 acts via another mechanism. [source] G protein-independent neuromodulatory action of adenosine on metabotropic glutamate signalling in mouse cerebellar Purkinje cellsTHE JOURNAL OF PHYSIOLOGY, Issue 2 2007Toshihide Tabata Adenosine receptors (ARs) are G protein-coupled receptors (GPCRs) mediating the neuromodulatory actions of adenosine that influence emotional, cognitive, motor, and other functions in the central nervous system (CNS). Previous studies show complex formation between ARs and metabotropic glutamate receptors (mGluRs) in heterologous systems and close colocalization of ARs and mGluRs in several central neurons. Here we explored the possibility of intimate functional interplay between Gi/o protein-coupled A1 -subtype AR (A1R) and type-1 mGluR (mGluR1) naturally occurring in cerebellar Purkinje cells. Using a perforated-patch voltage-clamp technique, we found that both synthetic and endogenous agonists for A1R induced continuous depression of a mGluR1-coupled inward current. A1R agonists also depressed mGluR1-coupled intracellular Ca2+ mobilization monitored by fluorometry. A1R indeed mediated this depression because genetic depletion of A1R abolished it. Surprisingly, A1R agonist-induced depression persisted after blockade of Gi/o protein. The depression appeared to involve neither the cAMP-protein kinase A cascade downstream of the alpha subunits of Gi/o and Gs proteins, nor cytoplasmic Ca2+ that is suggested to be regulated by the beta-gamma subunit complex of Gi/o protein. Moreover, A1R did not appear to affect Gq protein which mediates the mGluR1-coupled responses. These findings suggest that A1R modulates mGluR1 signalling without the aid of the major G proteins. In this respect, the A1R-mediated depression of mGluR1 signalling shown here is clearly distinguished from the A1R-mediated neuronal responses described so far. These findings demonstrate a novel neuromodulatory action of adenosine in central neurons. [source] Complex interplay between glutamate receptors and intracellular Ca2+ stores during ischaemia in rat spinal cord white matterTHE JOURNAL OF PHYSIOLOGY, Issue 1 2006Mohamed Ouardouz Electrophysiological recordings of propagated compound action potentials (CAPs) and axonal Ca2+ measurements using confocal microscopy were used to study the interplay between AMPA receptors and intracellullar Ca2+ stores in rat spinal dorsal columns subjected to in vitro combined oxygen and glucose deprivation (OGD). Removal of Ca2+ or Na+ from the perfusate was protective after 30 but not 60 min of OGD. TTX was ineffective with either exposure, consistent with its modest effect on ischaemic depolarization. In contrast, AMPA antagonists were very protective, even after 60 min of OGD where 0Ca2++ EGTA perfusate was ineffective. Similarly, blocking ryanodine receptor-mediated Ca2+ mobilization from internal stores (0Ca2++ nimodipine or 0Ca2++ ryanodine), or inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ release (block of group 1 metabotropic glutamate receptors with 1-aminoindan-1,5-dicarboxylic acid, inhibition of phospholipase C with U73122 or IP3 receptor block with 2APB; each in 0Ca2+) were each very protective, with the combination resulting in virtually complete functional recovery after 1 h OGD (97 ± 32% CAP recovery versus 4 ± 6% in artificial cerebrospinal fluid). AMPA induced a rise in Ca2+ concentration in normoxic axons, which was greatly reduced by blocking ryanodine receptors. Our data therefore suggest a novel and surprisingly complex interplay between AMPA receptors and Ca2+ mobilization from intracellular Ca2+ stores. We propose that AMPA receptors may not only allow Ca2+ influx from the extracellular space, but may also significantly influence Ca2+ release from intra-axonal Ca2+ stores. In dorsal column axons, AMPA receptor-dependent mechanisms appear to exert a greater influence than voltage-gated Na+ channels on functional outcome following OGD. [source] Pharmacological characterization of a Bombyx mori ,-adrenergic-like octopamine receptor stably expressed in a mammalian cell lineARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 2 2010Jia Huang Abstract Series of agonists and antagonists were examined for their actions on a Bombyx mori,-adrenergic-like octopamine receptor (OAR) stably expressed in HEK-293 cells. The rank order of potency of the agonists was clonidine>naphazoline>tolazoline in Ca2+ mobilization assays, and that of the antagonists was chlorpromazine>yohimbine. These findings suggest that the B. mori OAR is more closely related to the class-1 OAR in the intact tissue than to the other classes. N,-(4-Chloro- o -tolyl)- N -methylformamidine (DMCDM) and 2-(2,6-diethylphenylimino)imidazolidine (NC-5) elevated the intracellular calcium concentration ([Ca2+]i) with EC50s of 92.8,µM and 15.2,nM, respectively. DMCDM and NC-5 led to increases in intracellular cAMP concentration ([cAMP]i) with EC50s of 234,nM and 125,nM, respectively. The difference in DMCDM potencies between the cAMP and Ca2+ assays might be due to "functional selectivity." The Ca2+ and cAMP assay results for DMCDM suggest that the elevation of [cAMP]i, but not that of [Ca2+]i, might account for the insecticidal effect of formamidine insecticides. © 2009 Wiley Periodicals, Inc. [source] Different Ca2+ signalling cascades manifested by mastoparan in the prothoracic glands of the tobacco hornworm, Manduca sexta, and the silkworm, Bombyx moriARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 2 2007Skarlatos G. Dedos Abstract Application of the tetradecapeptide mastoparan to the prothoracic glands (PGs) of the tobacco hornworm, Manduca sexta, and the silkworm, Bombyx mori, resulted in increases in intracellular Ca2+ ([Ca2+]i). In M. sexta, Gi proteins are involved in the mastoparan-stimulated increase in [Ca2+]i. However, there is no involvement of Gi proteins in the mastoparan-stimulated increase in [Ca2+]i in prothoracic gland cells from B. mori. Unlike in M. sexta prothoracic glands, in B. mori prothoracic glands mastoparan increases [Ca2+]i even in the absence of extracellular Ca2+. Pharmacological manipulation of the Ca2+ signalling cascades in the prothoracic glands of both insect species suggests that in M. sexta prothoracic glands, mastoparan's first site of action is influx of Ca2+ through plasma membrane Ca2+ channels while in B. mori prothoracic glands, mastoparan's first site of action is mobilization of Ca2+ from intracellular stores. In M. sexta, the combined results indicate the presence of mastoparan-sensitive plasma membrane Ca2+ channels, distinct from those activated by prothoracicotropic hormone or the IP3 signalling cascade, that coordinate spatial increases in [Ca2+]i in prothoracic gland cells. We propose that in B. mori, mastoparan stimulates Ca2+ mobilization from ryanodine-sensitive intracellular Ca2+ stores in prothoracic gland cells. Arch. Insect Biochem. Physiol. 65:52,64, 2007. © 2007 Wiley-Liss, Inc. [source] Lindane (,-Hexachlorocyclohexane) Induces Internal Ca2+ Release and Capacitative Ca2+ Entry in Madin-Darby Canine Kidney CellsBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 4 2000Cheng-Hsien Lu The effect of lindane (,-hexachlorocyclohexane), an organochlorine pesticide, on Ca2+ mobilization in Madin-Darby canine kidney cells was examined by fluorimetry using fura-2 as a Ca2+ indicator. Lindane (5,200 ,M) increased [Ca2+]i concentration-dependently. The [Ca2+]i signal comprised an immediate initial rise followed by a persistent phase. Ca2+ removal inhibited the [Ca2+]i signal by reducing both the initial rise and the sustained phase. This implies lindane-triggered Ca2+ influx and Ca2+ release. In Ca2+ -free medium, 0.15 mM lindane increased [Ca2+]i after pretreatment with carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 ,M), a mitochondrial uncoupler, and two endoplasmic reticulum Ca2+ pump inhibitors, thapsigargin and cyclopiazonic acid. Conversely, pretreatment with lindane abolished CCCP- and thapsigargin-induced Ca2+ release. This suggests that 0.15 mM lindane released Ca2+ from the endoplasmic reticulum, mitochondria and other stores. La3+ (1 mM) partly inhibited 0.1 mM lindane-induced [Ca2+]i increase, confirming that lindane induced Ca2+ influx. Addition of 3 mM Ca2+ increased [Ca2+]i after pretreatment with 0.15 mM lindane for 750 sec. in Ca2+ -free medium, which indicates lindane-induced capacitative Ca2+ entry. Lindane (0.15 mM)-induced Ca2+ release was not reduced by inhibiting phospholipase C with 2 ,M U73122, but was inhibited by 70% by the phospholipase A2 inhibitor aristolochic acid (40 ,M). [source] Cathelicidin LL-37 induces the generation of reactive oxygen species and release of human ,-defensins from neutrophilsBRITISH JOURNAL OF DERMATOLOGY, Issue 6 2007Y. Zheng Summary Background, Psoriasis is characterized by epidermal infiltration of neutrophils that destroy invading microorganisms via a potent antimicrobial arsenal of oxidants and antimicrobial agents. In contrast to atopic dermatitis, psoriasis exhibits low levels of skin infections due to the presence of antimicrobial agents, including cathelicidin LL-37. LL-37 kills a broad spectrum of microbes, and activates neutrophil chemotaxis. Objective, To determine whether or not LL-37 could regulate additional neutrophil functions such as production of cytokines/chemokines, reactive oxygen species and release of neutrophil antimicrobial peptides. Methods, Human peripheral blood neutrophils were used in this study. The production of interleukin (IL)-8 and release of ,-defensins were analysed by enzyme-linked immunosorbent assay, and real-time polymerase chain reaction (PCR) was used to quantify ,-defensin gene expression. Phosphorylation of mitogen-activated protein kinase (MAPK) was determined by Western blotting. The generation of reactive oxygen species was examined using flow cytometry, and intracellular Ca2+ mobilization was measured using a calcium assay kit. Results, LL-37 enhanced the production of IL-8 under the control of MAPK p38 and extracellular signal regulated kinase (ERK), as evidenced by the inhibitory effects of p38 and ERK1/2 inhibitors on LL-37-mediated IL-8 production. Furthermore, LL-37 induced phosphorylation of p38 and ERK. We also revealed that LL-37 stimulated the generation of reactive oxygen species dose- and time-dependently, most probably via NADPH oxidase activation and intracellular Ca2+ mobilization. Finally, LL-37 induced both mRNA expression and protein release of ,-defensins, known as human neutrophil peptide 1,3. Conclusion, Taken together, we suggest that in addition to its microbicidal properties, LL-37 may contribute to innate immunity by enhancing neutrophil host defence functions at inflammation and/or infection sites. [source] | ||||||||||||||||