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Intracellular Calcium (intracellular + calcium)
Terms modified by Intracellular Calcium Selected AbstractsNANOMOLAR LEVEL OF OUABAIN INCREASES INTRACELLULAR CALCIUM TO PRODUCE NITRIC OXIDE IN RAT AORTIC ENDOTHELIAL CELLSCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 5-6 2004Xian Hui Dong Summary 1.,Changes in [Ca2+]i across the cell membrane and/or the sarcoplasmic reticulum regulate endothelial nitric oxide (NO) synthase activity. 2.,In the present study, we investigated the effect of ouabain, a specific inhibitor of Na+/K+ -ATPase, on NO release and [Ca2+]i movements in cultured rat aortic endothelial cells (RAEC) by monitoring NO production continuously using an NO-specific real-time sensor and by measuring the change in [Ca2+]i using a fluorescence microscopic imaging technique with high-speed wavelength switching. The t˝ (half-time of the decline of [Ca2+]i to basal levels after stimulation with 10 µmol/L bradykinin) was used as an index of [Ca2+]i extrusion. 3.,A very low concentration of ouabain (10 nmol/L) did not increase the peak of NO production, but decreased the decay of NO release and, accordingly, increased integral NO production by the maximal dose,response concentration induced by bradykinin. The same dose of ouabain affected [Ca2+]i movements across the cell membrane and/or sarcoplasmic reticulum induced by bradykinin with a time-course similar to that of NO release. Moreover, the t˝ was significantly increased. 4.,Pretreatment of RAEC with Na+ -free solution, an inhibitor of the Na+/Ca2+ exchanger, and nickel chloride hexahydrate prevented the effects induced by bradykinin and ouabain. 5.,These observations using real-time recording indicate that a small amount of ouabain contributes to the bradykinin-stimulated increase of NO production through inhibition of plasma membrane Na+/K+ -ATPase activity and an increase in intracellular Na+ concentrations. The membrane was then depolarized, leading to a decline in the bradykinin-stimulated increase in [Ca2+]i by forward mode Na+/Ca2+ exchange to prolong the Ca2+ signal time. 6.,From these results, we suggest that nanomolar levels of ouabain modulate [Ca2+]i movements and NO production in RAEC. [source] Simultaneous Optical Mapping of Transmembrane Potential and Intracellular Calcium in Myocyte CulturesJOURNAL OF CARDIOVASCULAR ELECTROPHYSIOLOGY, Issue 5 2000VLADIMIR G. FAST Ph.D. Simultaneous Mapping of Vm and Cai2+. Introduction: Fast spatially resolved measurements of transmembrane potential (Vm) and intracellular calcium (Cai2+) are important for studying mechanisms of arrhythmias and defibrillation. The goals of this work were (1) to develop an optical technique for simultaneous multisite optical recordings of Vm and Cai2+, and (2) to determine the relationship between Vm and Cai2+ during normal impulse propagation in myocyte cultures. Methods and Results: Monolayers of neonatal rat myocytes were stained with fluorescent dye RH-237 (Vm) and Fluo-3AM (Cai2+). Both dyes were excited at the same wavelength range. The emitted fluorescent was optically separated into components corresponding to changes in Vm, and Cai2+ and measured using two 16 × 16 photodiode arrays at a spatial resolution of up to 27.5 ,m per diode and sampling rate of 2.5 kHz. The optical setup was adjusted so that there was no optical cross-talk between the two types of measurements, which was validated in experiments involving staining with either RH-237 or Fluo-3. The amplitude of Fluo-3 signals rapidly decreased during experiments due to dye leakage. Dye leakage was substantially reduced by application of 1 mM probenecid, a blocker of organic anion transport, which had no effect on action potential duration and only minor effect on conduction velocity. In double-stained preparations, during regular pacing Cai2+ transients had a rise time of 14.2 ± 2 msec, and they followed Vm upstrokes with a delay of 5.3 ± 1 msec (n = 9). Durations of Vm, and Cai2+ transients determined at 50% level of signal recovery were 54.6 ± 10 msec and 136 ± 8 msec, respectively. Application of 2 ,M nifedipine reduced the amplitude and duration of Cai2+ transients without significantly affecting conduction velocity. Conclusion: The results demonstrate feasibility of simultaneous optical recordings of Vm and Cai2+ transients with high spatial and temporal resolution. [source] Effects of Low Power Laser Irradiation on Intracellular Calcium and Histamine Release in RBL-2H3 Mast CellsPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2007Wen-Zhong Yang Although laser irradiation has been reported to promote skin wound healing, the mechanism is still unclear. As mast cells are found to accumulate at the site of skin wounds we hypothesized that mast cells might be involved in the biological effects of laser irradiation. In this work the mast cells, RBL-2H3, were used in vitro to investigate the effects of laser irradiation on cellular responses. After laser irradiation, the amount of intracellular calcium ([Ca2+]i) was increased, followed by histamine release, as measured by confocal fluorescence microscopy with Fluo-3/AM staining and a fluorescence spectrometer with o -phthalaldehyde staining, respectively. The histamine release was mediated by the increment of [Ca2+]i from the influx of the extracellular buffer solution through the cation channel protein, transient receptor potential vanilloid 4 (TRPV4). The TRPV4 inhibitor, Ruthenium Red (RR) can effectively block such histamine release, indicating that TRPV4 was the key factor responding to laser irradiation. These induced responses of mast cells may provide an explanation for the biological effects of laser irradiation on promoting wound healing, as histamine is known to have multi-functions on accelerating wound healing. [source] Melatonin Counteracts Alterations in Oxidative Metabolism and Cell Viability Induced by Intracellular Calcium Overload in Human Leucocytes: Changes with AgeBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 1 2010Javier Espino In fact, the free radical theory of ageing proposes that deleterious actions of free radicals are responsible for the functional deterioration associated with ageing. Moreover, a close relationship exists between calcium homeostasis and oxidative stress. The current work was aimed at proving that intracellular calcium overload induced by N -formyl-methionyl-leucyl-phenylalanine (FMLP) and/or thapsigargin leads to oxidative stress. We additionally examined the effect of melatonin on the levels of reactive oxygen species (ROS) and cell viability in human leucocytes collected from young (20,30-year-old) and elderly (65,75-year-old) individuals under both basal and oxidative stress-induced conditions. Treatments with 10 nM FMLP and/or 1 ,M thapsigargin induced a transient increase in cytosolic free-calcium concentration ([Ca2 + ]c) in human leucocytes due to calcium release from internal stores, and led in turn to oxidative stress, as assessed by intracellular ROS measurement. Non-treated leucocytes from aged individuals exhibited higher ROS levels and lower rates of cell survival when compared to leucocytes from young individuals. Similar results were obtained in FMLP and/or thapsigargin-treated leucocytes from elderly individuals when compared to those from the young individuals. Melatonin treatment significantly reduced both hydrogen peroxide (H2O2) and superoxide anion levels, likely due to its free-radical scavenging properties, and enhanced leucocyte viability in both age groups. Therefore, melatonin may be a useful tool for the treatment of disease states and processes where an excessive production of oxidative damage occurs. [source] Mechanisms Associated with the Negative Inotropic Effect of Deuterium Oxide in Single Rat Ventricular MyocytesEXPERIMENTAL PHYSIOLOGY, Issue 2 2000K. Hongo Deuterium oxide (D2O) is known to cause a negative inotropic effect in muscle although the mechanisms associated with this response in cardiac muscle are not well understood. We studied the effects of D2O in single rat ventricular myocytes in order to characterise the mechanisms associated with its negative inotropic effect and to assess its possible use as an acute modulator of microtubules. D2O rapidly reduced the magnitude of contraction in rat ventricular myocytes, and there was some recovery of contraction in the presence of D2O. Colchicine, an agent known to depolymerise microtubules, did not modify the effect of D2O. D2O decreased the L-type Ca2+ current (ICa), measured under whole cell and perforated patch clamp conditions. Slowing of the time to peak and a delay in inactivation of ICa were observed. Intracellular calcium ([Ca2+]i) and sodium ([Na+]i) were measured using the fluorescent indicators fura-2 and SBFI, respectively. The fall in contraction upon exposure to D2O was not associated with a fall in the [Ca2+]i transient; this response is indicative of a reduction in myofilament Ca2+ sensitivity. Both the [Ca2+]i transient and [Na+]i increased during the partial recovery of contraction in the presence of D2O. We conclude that a decrease in the myofilament sensitivity for Ca2+ and a reduction in Ca2+ influx via ICa are principally responsible for the negative inotropic effect of D2O in cardiac muscle. We found no evidence to explain the negative inotropic effect of D2O in terms of microtubule proliferation. In addition we suggest that acute application of D2O is not a useful procedure for the investigation of the role of microtubules in excitation-contraction coupling in cardiac muscle. [source] Changes in oxidative balance in rat pericytes exposed to diabetic conditionsJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 1 2004A. Manea Abstract Recent data indicate that the oxidative stress plays an important role in the pathogenesis of diabetes and its complications such as retinopathy, nephropathy and accelerated atherosclerosis. In diabetic retinopathy, it was demonstrated a selective loss of pericytes accompanied by capillary basement membrane thickening, increased permeability and neovascularization. This study was designed to investigate the role of diabetic conditions such as high glucose, AGE-Lysine, and angiotensin II in the modulation of antioxidant enzymes activities, glutathione level and reactive oxygen species (ROS) production in pericytes. The activity of antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and total glutathione (GSH) was measured spectrophotometrically. The production of ROS was detected by spectrofluorimetry and fluorescence microscopy after loading the cells with 2,-7, dichlorofluoresceine diacetate; as positive control H2O2 was used. Intracellular calcium was determined using Fura 2 AM assay. The results showed that the cells cultured in high glucose alone, do not exhibit major changes in the antioxidant enzyme activities. The presence of AGE-Lys or Ang II induced the increase of SOD activity. Their combination decreased significantly GPx activity and GSH level. Athree times increase in ROS production and a significant impairment of intracellular calcium homeostasis was detected in cells cultured in the presence of the three pro-diabetic agents used. In conclusion, our data indicate that diabetic conditions induce in pericytes: (i) an increase of ROS and SOD activity, (ii) a decrease in GPx activity and GSH level, (iii) a major perturbation of the intracellular calcium homeostasis. The data may explain the structural and functional abnormalities of pericytes characteristic for diabetic retinopathy. [source] SERCA function declines with age in adrenergic nerves from the superior cervical ganglionAUTONOMIC & AUTACOID PHARMACOLOGY, Issue 5-6 2000W. J. Pottorf 1 Intracellular calcium is a universal second messenger integrating numerous cellular pathways. An age-related breakdown in the mechanisms controlling [Ca2+]i homeostasis could contribute to neuronal degeneration. One component of neuronal calcium regulation believed to decline with age is the function of sarco/endoplasmic reticulum calcium ATPase (SERCA) pumps. 2 Therefore we investigated the impact of age on the capacity of SERCA pumps to control high (68 m M) [K+]-evoked [Ca2+]i -transients in acutely dissociated superior cervical ganglion (SCG) cells from 6- and 20-month-old Fisher-344 rats. Calcium transients were measured by fura-2 microfluorometry in the presence of vanadate (0.1 ,M) to selectively block plasma membrane calcium ATPase (PMCA) pumps, dinitrophenol (100 ,M) to block mitochondrial calcium uptake and extracellular sodium replaced with tetraethylammonium to block Na+/Ca2+ -exchanger, thus forcing the neuronal cells to rely on SERCA uptake to control [Ca2+]i homeostasis. 3 In the presence of these calcium buffering blockers, the rate of recovery of [Ca2+]i was significantly slower and time to recover to approximately 90% of resting [Ca2+]i was significantly greater in SCG cells from old (20 months) compared with young (6 months) animals. 4 This age-related change in the recovery phase of [K+]-evoked [Ca2+]i -transients could not be explained by differences in the sensitivity of SCG cells to the calcium buffering blockers, as no age-related difference in basal [Ca2+]i was observed. 5 These studies illustrate that when rat SCG cells are forced to rely on SERCAs to buffer [K+]-evoked [Ca2+]i -transients, an age-related decline in SERCA function is revealed. Such age-related declines in calcium regulation coupled with neuronal sensitivity to calcium overload underscore the importance of understanding the components of [Ca2+]i homeostasis and the functional compensation that may occur with advancing age. [source] Osteopontin Mediates Dense Culture-Induced Proliferation and Adhesion of Prostate Tumour Cells: Role of Protein Kinase C, p38 Mitogen-Activated Protein Kinase and CalciumBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 2 2009Hong Zhou Our results demonstrate that increasing cell density exerted stress on PC-3M cells, which decreased cell proliferation in dense cultures, but tended to facilitate tumour metastasis since cell adhesion ability was elevated and the cells showed an increased growth rate after being moved to a favourable growth environment. We conclude that higher cell density-mediated pericellular hypoxia was an important factor inducing expression of the intrinsic hypoxia marker osteopontin, another mechanism contributing to cell adhesion enhancement in PC-3M cells. In addition, cell density enhanced adhesion ability due to the activation of p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase C. Intracellular calcium also played positive roles at least partially through activating p38 MAPK. [source] Effects of short-term food deprivation on orexin-A-induced intestinal bicarbonate secretion in comparison with related secretagoguesACTA PHYSIOLOGICA, Issue 3 2010G. Flemström Abstract Studies of gastrointestinal physiology in humans and intact animals are usually conducted after overnight fast. We compared the effects of orexin-A, vasoactive intestinal polypeptide (VIP), melatonin, serotonin, uroguanylin, ghrelin and prostaglandin E2 (PGE2) on duodenal bicarbonate secretion in fed and overnight fasted animals. This review is a summary of our findings. Secretagogues were administered by intra-arterial infusion or luminally (PGE2). Enterocyte intracellular calcium ([Ca2+]i) signalling was studied by fluorescence imaging. Total RNA was extracted, reverse transcripted to cDNA and expression of orexin receptors measured by quantitative real-time PCR. Orexin-A stimulates the duodenal secretion in continuously fed animals but not in food-deprived animals. Similarly, short-term fasting causes a 100-fold decrease in the amount of the muscarinic agonist bethanechol required for stimulation of secretion. In contrast, fasting does not affect secretory responses to intra-arterial VIP, melatonin, serotonin, uroguanylin and ghrelin, or that to luminal PGE2. Orexin-A induces [Ca2+]i signalling in enterocytes from fed rats but no significant [Ca2+]i responses occur in enterocytes from fasted animals. In addition, overnight fasting decreases the expression of mucosal orexin receptors. Short-term food deprivation thus decreases duodenal expression of orexin receptors and abolishes the secretory response to orexin-A as well as orexin-A-induced [Ca2+]i signalling. Fasting, furthermore, decreases mucosal sensitivity to bethanechol. The absence of declines in secretory responses to other secretagogues tested strongly suggests that short-term fasting does not affect the secretory capacity of the duodenal mucosa in general. Studies of intestinal secretion require particular evaluation with respect to feeding status. [source] Angiotensin II enhances the afferent arteriolar response to adenosine through increases in cytosolic calciumACTA PHYSIOLOGICA, Issue 4 2009E. Y. Lai Abstract Aims:, Angiotensin II (Ang II) is a strong renal vasoconstrictor and modulates the tubuloglomerular feedback (TGF). We hypothesized that Ang II at low concentrations enhances the vasoconstrictor effect of adenosine (Ado), the mediator of TGF. Methods:, Afferent arterioles of mice were isolated and perfused, and both isotonic contractions and cytosolic calcium transients were measured. Results:, Bolus application of Ang II (10,12 and 10,10 m) induced negligible vasoconstrictions, while Ang II at 10,8 m reduced diameters by 35%. Ang II at 10,12, 10,10 and 10,8 m clearly enhanced the arteriolar response to cumulative applications of Ado (10,11 to 10,4 m). Ado application increased the cytosolic calcium concentrations in the vascular smooth muscle, which were higher at 10,5 m than at 10,8 m. Ang II (10,11 to 10,6 m) also induced concentration-dependent calcium transients, which were attenuated by AT1 receptor inhibition. Simultaneously applied Ang II (10,10 m) additively enhanced the calcium transients induced by 10,8 and 10,5 m Ado. The transients were partly inhibited by AT1 or A1 receptor antagonists, but not significantly by A2 receptor antagonists. Conclusion:, A low dose of Ang II enhances Ado-induced constrictions, partly via AT1 receptor-mediated calcium increase. Ado increases intracellular calcium by acting on A1 but not A2 receptors. The potentiating effect of Ang II on Ado-induced arteriolar vasoconstrictions may involve calcium sensitization of the contractile machinery, as Ang II only additively increased cytosolic calcium concentrations, while its effect on the arteriolar constriction was more than additive. The potentiating effect of Ang II might contribute to the resetting of TGF. [source] Dopamine modulation of the In vivo acetylcholine response in the Drosophila mushroom bodyDEVELOPMENTAL NEUROBIOLOGY, Issue 11 2009Vitold Tsydzik Abstract Olfactory sensory information in Drosophila is transmitted through antennal lobe projections to Mushroom Body neurons (Kenyon cells) by means of cholinergic synapses. Application of acetylcholine (ACh) and odors produce significant increases in intracellular calcium ([Ca2+]i) in these neurons. Behavioral studies show that Kenyon cell activity is modulated by dopaminergic inputs and this modulation is thought to be the basis for an olfactory conditioned response. However, quantitative assessment of the synaptic inputs to Kenyon cells is currently lacking. To assess neuronal activity under in vivo conditions, we have used the endogenously-expressed camgaroo reporter to measure [Ca2+]i in these neurons. We report here the dose-response relationship of Kenyon cells for ACh and dopamine (DA). Importantly, we also show that simultaneous application of ACh and DA results in a significant decrease in the response to ACh alone. In addition, we show inhibition of the ACh response by cyclic adenosine monophosphate. This is the first quantitative assessment of the effects of these two important transmitters in this system, and it provides an important basis for future analysis of the cellular mechanisms of this well established model for associative olfactory learning. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009 [source] Widely distributed Drosophila G-protein-coupled receptor (CG7887) is activated by endogenous tachykinin-related peptidesDEVELOPMENTAL NEUROBIOLOGY, Issue 1 2006Ryan T. Birse Abstract Neuropeptides related to vertebrate tachykinins have been identified in Drosophila. Two Drosophila G-protein-coupled receptors (GPCRs), designated NKD (CG6515) and DTKR (CG7887), cloned earlier, display sequence similarities to mammalian tachykinin receptors. However, they were not characterized with the endogenous Drosophila tachykinins (DTKs). The present study characterizes one of these receptors, DTKR. We determined that HEK-293 cells transfected with DTKR displayed dose-dependent increases in both intracellular calcium and cyclic AMP levels in response to the different DTK peptides. DTK peptides also induced internalization of DTKR-green fluorescent protein (GFP) fusion constructs in HEK-293 cells. We generated specific antireceptor antisera and showed that DTKR is widely distributed in the adult brain and more scarcely in the larval CNS. The distribution of the receptor in brain neuropils corresponds well with the distribution of its ligands, the DTKs. Our findings suggest that DTKR is a DTK receptor in Drosophila and that this ligand-receptor system plays multiple functional roles. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2006 [source] Further studies on the interaction of loperamide with capacitative calcium entry in Leukemic HL-60 cells,DRUG DEVELOPMENT RESEARCH, Issue 11 2006John W. Daly Abstract Loperamide at 3,10,µM has augmentative effects on calcium levels elevated by capacitative calcium entry (CCE) in leukemic HL-60 cells after release of intracellular calcium by ATP or thapsigargin (Harper et al. [1997] Proc Natl Acad Sci USA 94:14912,14917). The effect of loperamide on calcium levels was absent at a pH value of 6.8, a pH at which CCE is not active in HL-60 cells. Further investigations of HL-60 cells in recent years revealed a great reduction in the magnitude of the loperamide response. However, when preceded by a CCE blocker, namely N-methylnitrendipine (MRS 1844) or N-propargylnitrendipine (MRS 1845), loperamide caused a significant reversal of the blockade. Six structural analogs of loperamide were synthesized, but only two showed loperamide-like activity. Drug Dev. Res. 67:842,851, 2006. Published 2007 Wiley-Liss, Inc. [source] Expression of multiple P2Y receptors by MDCK-D1 cells: P2Y1 receptor cloning and signalingDRUG DEVELOPMENT RESEARCH, Issue 1 2003Richard J. Hughes The Madin Darby canine kidney (MDCK) cell line, a well-differentiated renal epithelial cell line, is a useful model to examine P2Y receptor signaling and response. Our studies with MDCK-D1, a clonal isolate, demonstrate that these cells release ATP in response to mechanical stimulation and activation of certain G-protein-coupled receptors. Reverse transcriptase-polymerase chain reaction (RT-PCR) studies document that MDCK cells express multiple P2Y receptors, including P2Y1, P2Y2, P2Y6, and P2Y11 receptors. We isolated cDNAs for several of the P2Y receptor genes and expressed these in cells, such as the 1321N1 astrocytoma cell line, that lack native P2Y receptor expression. We report here the molecular cloning of the MDCK P2Y1 receptor, heterologous expression in 1321N1 cells, and the ability of the heterologously expressed receptors to increase intracellular calcium and phosphoinositide hydrolysis. ADP, methylthioATP, and ADP,S are agonists with the greatest potency, while ATP and ATP,S show lower potency and efficacy, and benzoylbenzoylATP, UTP, and UDP lack efficacy at the cloned P2Y1 receptor. Several antagonists, including MRS2179, A3P5PS, suramin, and PPADS blocked response at the cloned P2Y1 receptors. With their ability to respond to ADP and ATP, P2Y1 receptors, along with other P2Y receptors expressed in MDCK cells, contribute to the response of these cells to ATP (or its breakdown product, ADP) released from the cells and to exogenously added nucleotides. Drug Dev. Res. 59:1,7, 2003. © 2003 Wiley-Liss, Inc. [source] Phospholipase,C, negatively regulates Rac/Cdc42 activation in antigen-stimulated mast cellsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 1 2007Mirvat El-Sibai M.D. Abstract The Rho GTPases Rac and Cdc42 play a central role in the regulation of secretory and cytoskeletal responses in antigen-stimulated mast cells. In this study, we examine the kinetics and mechanism of Rac and Cdc42 activation in the rat basophilic leukemia RBL-2H3 cells. The activation kinetics of both Rac and Cdc42 show a biphasic profile, consisting of an early transient peak at 1,min and a late sustained activation phase at 20,40,min. The inhibition of phospholipase,C (PLC), causes a twofold increase in Rac and Cdc42 activation that coincides with a dramatic production of atypical filopodia-like structures. Inhibition of protein kinase,C using bisindolylmaleimide mimics the effect of PLC, inhibition on Rac activation, but not on Cdc42 activation. In contrast, depletion of intracellular calcium leads to a complete inhibition of the early activation peak of both Rac and Cdc42, without significant effects on the late sustained activation. These data suggest that PLC, is involved in a negative feedback loop that leads to the inhibition of Rac and Cdc42. They also suggest that the presence of intracellular calcium is a prerequisite for both Rac and Cdc42 activation. [source] CD46-mediated costimulation induces a Th1-biased response and enhances early TCR/CD3 signaling in human CD4+ T,lymphocytesEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 9 2004Alejandra Sánchez Abstract The role of membrane cofactor protein (MCP, CD46) on human T,cell activation has been analyzed. Coligation of CD3 and CD46 in the presence of PMA or CD28 costimuli enhanced IL-2, IFN-,, or IL-10 secretion by CD4+ T,lymphocytes. The effect of CD46 on IL-10 secretion did not require additional costimuli like anti-CD28 antibodies or phorbol esters. CD46 also enhanced IL-2 or IFN-, secretion by CD4+ blasts. In contrast, IL-5 secretion was inhibited upon CD46-CD3 coligation, in all the cells analyzed. These effects were independent of IL-12 and suggest that CD46 costimulation promotes a Th1-biased response in human CD4+ T,lymphocytes. CD46 enhanced TCR/CD3-induced tyrosine phosphorylation of CD3, and ZAP-70, as well as the activation of the ERK, JNK, and p38, but did not modify intracellular calcium. The effect of specific inhibitors shows that enhanced ERK activation contributes to augmented IFN-, and lower IL-5 secretion and, consequently, to the Th1 bias. Cross-linking CD46 alone induced weak tyrosine phosphorylation of CD3, and ZAP-70. However, CD46 cross-linking by itself did not induce cell proliferation or lymphokine secretion, and pretreatment of CD4+ T,lymphocytes with anti-CD46 antibodies did not significantly alter TCR/CD3 activation. [source] Characterization of a novel NCAM ligand with a stimulatory effect on neurite outgrowth identified by screening a combinatorial peptide libraryEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 9 2002Lars C. B. Rřnn 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] The retrograde inhibition of IPSCs in rat cerebellar Purkinje cells is highly sensitive to intracellular Ca2+EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2000Maike Glitsch Abstract The Ca2+ -dependent retrograde inhibition of inhibitory postsynaptic currents (depolarization-induced-suppression of inhibition; DSI) was investigated using fura-2 Ca2+ measurements and whole-cell patch-clamp recordings in rat cerebellar Purkinje cells. DSI was studied in cells loaded with different concentrations of the Ca2+ chelators BAPTA and EGTA. A concentration of 40 m m BAPTA was required to significantly interfere with DSI, whereas 10 m m BAPTA was almost ineffective. 40 m m EGTA reduced DSI, but was less effective than 40 m m BAPTA. Ratiometric Ca2+ measurements indicated that the extent of DSI depended critically on the changes in intracellular calcium ([Ca2+]i). The relationship between DSI and peak ,[Ca2+]i could be approximated by a hyperbolic function, with apparent half-saturation concentrations of 200 and 40 n m for dendritic and somatic [Ca2+]i, respectively. It is suggested that DSI is due to somatodendritic exocytosis of a retrograde messenger, and that this exocytosis is highly sensitive to [Ca2+]i. [source] Antioxidant and anti-inflammatory activities of melanocortin peptidesEXPERIMENTAL DERMATOLOGY, Issue 9 2004J. W. Haycock ,-Melanocyte-stimulating hormone (,-MSH) has previously been identified as a potent anti-inflammatory agent in various tissues including the skin. It operates by binding to the melanocortin-1 receptor (MC-1R) which results in the elevation of cyclic AMP. ,-MSH opposes the action of several proinflammatory cytokines including tumour necrosis factor-, (TNF-,). We have shown that ,-MSH can inhibit TNF-,-stimulated activation of nuclear factor-,B (NF-,B) in human cultured melanocytes, melanoma cells, keratinocytes, fibroblasts, Schwann cells and olfactory ensheathing cells. It also inhibits TNF-,-stimulated upregulation of intercellular adhesion molecule-1 (ICAM-1) in many of these cells and can inhibit peroxide-stimulated activation of glutathione peroxidase, suggesting an antioxidant role. ,-MSH is also able to stimulate intracellular calcium release in keratinocytes and fibroblasts (which do not readily show detectible cyclic AMP elevation) but only in the presence of PIA (an adenosine agonist). The carboxyl terminal tripeptides KPV/KP-D-V are reported to be the minimal sequences necessary to convey anti-inflammatory potential, but evidence on how they act is not fully known. Stable transfection of Chinese hamster ovary cells with MC-1R suggests that the KPV peptides operate by this receptor, at least by elevating intracellular calcium. Elevation of cyclic AMP by these tripeptides has not been detected in any cell type studied; however, calcium elevation can inhibit TNF-,-stimulated NF-,B activity (as for cyclic AMP). In conclusion, the MSH peptides convey anti-inflammatory and antioxidant activity in many cell types in skin and nerve, by counteracting proinflammatory cytokine signalling. The KPV peptides appear to act functionally via the MC-1R and can also elevate intracellular calcium. [source] Carbonyl cyanide m -chlorophenylhydrazone induced calcium signaling and activation of plasma membrane H+ -ATPase in the yeast Saccharomyces cerevisiaeFEMS YEAST RESEARCH, Issue 4 2008Michele B.P. Pereira Abstract The plasma membrane H+ -ATPase from Saccharomyces cerevisiae is an enzyme that plays a very important role in the yeast physiology. The addition of protonophores, such as 2,4-dinitrophenol (DNP) and carbonyl cyanide m -chlorophenylhydrazone (CCCP), also triggers a clear in vivo activation of this enzyme. Here, we demonstrate that CCCP-induced activation of the plasma membrane H+ -ATPase shares some similarities with the sugar-induced activation of the enzyme. Phospholipase C and protein kinase C activities are essential for this activation process while Gpa2p, a G protein involved in the glucose-induced activation of the ATPase, is not required. CCCP also induces a phospholipase C-dependent increase in intracellular calcium. Moreover, we show that the availability of extracellular calcium is required for CCCP stimulation of H+ -ATPase, suggesting a possible connection between calcium signaling and activation of ATPase. [source] Calcium/calcineurin signaling in primary cortical astrocyte cultures: Rcan1-4 and cyclooxygenase-2 as NFAT target genesGLIA, Issue 7 2008Andrea Canellada Abstract The calcineurin/nuclear factor of activated T cells (NFAT) signaling pathway mediates important cell responses to calcium, but its activity and function in astrocytes have remained unclear. We show that primary cortical astrocyte cultures express the regulatory and catalytic subunits of the phosphatase calcineurin as well as the calcium-regulated NFAT family members (NFATc1, c2, c3, and c4). NFATs are activated by calcium-mobilizing agents in astrocytes, and this activation is blocked by the calcineurin inhibitor cyclosporine A. Microarray screening identified cyclooxygenase-2 (Cox-2), which is implicated in brain injury, and Rcan 1-4, an endogenous calcineurin inhibitor, as genes up-regulated by calcineurin-dependent calcium signals in astrocytes. Mobilization of intracellular calcium with ionophore potently augments the promoter activity and mRNA and protein expression of Rcan 1-4 and Cox-2 induced by combined treatment with phorbol esters. Moreover, Rcan 1-4 expression is efficiently induced by calcium mobilization alone. For both the genes, the calcium signal component is dependent on calcineurin and is replicated by exogenous expression of a constitutively active NFAT, strongly suggesting that the calcium-induced gene activation is mediated by NFATs. Finally, we report that calcineurin-dependent expression of Cox-2 and Rcan 1-4 is induced by physiological calcium mobilizing agents, such as thrombin, agonists of purinergic and glutamate receptors, and L-type voltage-gated calcium channels. These findings provide insights into calcium-initiated gene transcription in astrocytes, and have implications for the regulation of calcium responses in astrocytes. © 2008 Wiley-Liss, Inc. [source] Nucleotide-mediated calcium signaling in rat cortical astrocytes: Role of P2X and P2Y receptorsGLIA, Issue 3 2003Marta Fumagalli Abstract ATP is the dominant messenger for astrocyte-to-astrocyte calcium-mediated communication. Definition of the exact ATP/P2 receptors in astrocytes and of their coupling to intracellular calcium ([Ca2+]i) has important implications for brain physiology and pathology. We show that, with the only exception of the P2X6 receptor, primary rat cortical astrocytes express all cloned ligand-gated P2X (i.e., P2X1,5 and P2X7) and G-protein-coupled P2Y receptors (i.e., P2Y1, P2Y2, P2Y4, P2Y6, and P2Y12). These cells also express the P2Y-like UDP-glucose receptor, which has been recently recognized as the P2Y14 receptor. Single-cell image analysis showed that only some of these receptors are coupled to [Ca2+]i. While ATP induced rapid and transient [Ca2+]i increases (counteracted by the P2 antagonists suramin, pyridoxal-phosphate-6-azophenyl-2,-4,-disulfonic acid and oxidized ATP), the P2X1/P2X3 agonist ,,meATP produced no changes. Conversely, the P2X7 agonist BzATP markedly increased [Ca2+]i; the presence and function of the P2X7 receptor was also confirmed by the formation of the P2X7 pore. ADP and 2meSADP also produced [Ca2+]i increases antagonized by the P2Y1 antagonist MRS2179. Some cells also responded to UTP but not to UDP. Significant responses to sugar-nucleotides were also detected, which represents the first functional response reported for the putative P2Y14 receptor in a native system. Based on agonist preference of known P2 receptors, we conclude that, in rat astrocytes, ATP-induced calcium rises are at least mediated by P2X7 and P2Y1 receptors; additional receptors (i.e., P2X2, P2X4, P2X5, P2Y2, P2Y4, and P2Y14) may also contribute. © 2003 Wiley-Liss, Inc. [source] Systemic infusion of angiotensin II exacerbates liver fibrosis in bile duct,ligated rats,HEPATOLOGY, Issue 5 2005Ramón Bataller Recent evidence indicates that the renin,angiotensin system (RAS) plays a major role in liver fibrosis. Here, we investigate whether the circulatory RAS, which is frequently activated in patients with chronic liver disease, contributes to fibrosis progression. To test this hypothesis, we increased circulatory angiotensin II (Ang II) levels in rats undergoing biliary fibrosis. Saline or Ang II (25 ng/kg/h) were infused into bile duct,ligated rats for 2 weeks through a subcutaneous pump. Ang II infusion increased serum levels of Ang II and augmented bile duct ligation,induced liver injury, as assessed by elevated liver serum enzymes. Moreover, it increased the hepatic concentration of inflammatory proteins (tumor necrosis factor , and interleukin 1,) and the infiltration of CD43-positive inflammatory cells. Ang II infusion also favored the development of vascular thrombosis and increased the procoagulant activity of tissue factor in the liver. Livers from bile duct,ligated rats infused with Ang II showed increased transforming growth factor ,1 content, collagen deposition, accumulation of smooth muscle ,-actin,positive cells, and lipid peroxidation products. Moreover, Ang II infusion stimulated phosphorylation of c-Jun and p42/44 mitogen-activated protein kinase and increased proliferation of bile duct cells. In cultured rat hepatic stellate cells (HSCs), Ang II (10,8 mol/L) increased intracellular calcium and stimulated reactive oxygen species formation, cellular proliferation and secretion of proinflammatory cytokines. Moreover, Ang II stimulated the procoagulant activity of HSCs, a newly described biological function for these cells. In conclusion, increased systemic Ang II augments hepatic fibrosis and promotes inflammation, oxidative stress, and thrombogenic events. (HEPATOLOGY 2005;41:1046,1055.) [source] Detection of endogenous lithium in neuropsychiatric disorders,a model for biological transmutationHUMAN PSYCHOPHARMACOLOGY: CLINICAL AND EXPERIMENTAL, Issue 1 2002Ravi Kumar Kurup Abstract The human hypothalmus produces an endogenous membrane Na+ -K+ ATPase inhibitor, digoxin. A digoxin induced model of cellular/neuronal quantal state and perception has been described by the authors. Biological transmutation has been described in microbial systems in the quantal state. The study focuses on the plasma levels of digoxin, RBC membrane Na+ -K+ ATPase activity, plasma levels of magnesium and lithium in neuropsychiatric and systemic disorders. Inhibition of RBC membrane Na+ -K+ ATPase activity was observed in most cases along with an increase in the levels of serum digoxin and lithium and a decrease in the level of serum Mg++. The generation of endogenous lithium would obviously occur due to biological transmutation from magnesium. Digoxin and lithium together can produce added membrane Na+ -K+ ATPase inhibition. The role of membrane Na+ -K+ ATPase inhibition in the pathogenesis of neuropsychiatric and systemic disorders is discussed. The inhibition of membrane Na+ -K+ ATPase can contribute to an increase in intracellular calcium and a decrease in magnesium, which can result in a defective neurotransmitter transport mechanism, mitochondrial dysfunction and apoptosis, defective golgi body function and protein processing dysfunction, immune dysfunction and oncogenesis. Copyright © 2002 John Wiley & Sons, Ltd. [source] Soman-induced seizures: limbic activity, oxidative stress and neuroprotective proteins,,JOURNAL OF APPLIED TOXICOLOGY, Issue S1 2001T. L. Pazdernik Abstract Soman, a potent acetylcholinesterase inhibitor, induces status epilepticus in rats followed by conspicuous neuropathology, most prominent in piriform cortex and the CA3 region of the hippocampus. Cholinergic seizures originate in striatal,nigral pathways and with fast-acting agents (soman) rapidly spread to limbic related areas and finally culminate in a full-blown status epilepticus. This leads to neurochemical changes, some of which may be neuroprotective whereas others may cause brain damage. Pretreatment with lithium sensitizes the brain to cholinergic seizures. Likewise, other agents that increase limbic hyperactivity may sensitize the brain to cholinergic agents. The hyperactivity associated with the seizure state leads to an increase in intracellular calcium, cellular edema and metal delocalization producing an oxidative stress. These changes induce the synthesis of stress-related proteins such as heat shock proteins, metallothioneins and heme oxygenases. We show that soman-induced seizures cause a depletion in tissue glutathione and an increase in tissue ,catalytic' iron, metallothioneins and heme oxygenase-1. The oxidative stress induces the synthesis of stress-related proteins, which are indicators of ,stress' and possibly provide neuroprotection. These findings suggest that delocalization of iron may catalyze Fenton-like reactions, causing progressive cellular damage via free radical products. Copyright © 2001 John Wiley & Sons, Ltd. [source] Simultaneous Optical Mapping of Transmembrane Potential and Intracellular Calcium in Myocyte CulturesJOURNAL OF CARDIOVASCULAR ELECTROPHYSIOLOGY, Issue 5 2000VLADIMIR G. FAST Ph.D. Simultaneous Mapping of Vm and Cai2+. Introduction: Fast spatially resolved measurements of transmembrane potential (Vm) and intracellular calcium (Cai2+) are important for studying mechanisms of arrhythmias and defibrillation. The goals of this work were (1) to develop an optical technique for simultaneous multisite optical recordings of Vm and Cai2+, and (2) to determine the relationship between Vm and Cai2+ during normal impulse propagation in myocyte cultures. Methods and Results: Monolayers of neonatal rat myocytes were stained with fluorescent dye RH-237 (Vm) and Fluo-3AM (Cai2+). Both dyes were excited at the same wavelength range. The emitted fluorescent was optically separated into components corresponding to changes in Vm, and Cai2+ and measured using two 16 × 16 photodiode arrays at a spatial resolution of up to 27.5 ,m per diode and sampling rate of 2.5 kHz. The optical setup was adjusted so that there was no optical cross-talk between the two types of measurements, which was validated in experiments involving staining with either RH-237 or Fluo-3. The amplitude of Fluo-3 signals rapidly decreased during experiments due to dye leakage. Dye leakage was substantially reduced by application of 1 mM probenecid, a blocker of organic anion transport, which had no effect on action potential duration and only minor effect on conduction velocity. In double-stained preparations, during regular pacing Cai2+ transients had a rise time of 14.2 ± 2 msec, and they followed Vm upstrokes with a delay of 5.3 ± 1 msec (n = 9). Durations of Vm, and Cai2+ transients determined at 50% level of signal recovery were 54.6 ± 10 msec and 136 ± 8 msec, respectively. Application of 2 ,M nifedipine reduced the amplitude and duration of Cai2+ transients without significantly affecting conduction velocity. Conclusion: The results demonstrate feasibility of simultaneous optical recordings of Vm and Cai2+ transients with high spatial and temporal resolution. [source] Vasomotion dynamics following calcium spiking depend on both cell signalling and limited constriction velocity in rat mesenteric small arteriesJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 3 2008Ed VanBavel Abstract Vascular smooth muscle cell contraction depends on intracellular calcium. However, calcium-contraction coupling involves a complex array of intracellular processes. Quantitating the dynamical relation between calcium perturbations and resulting changes in tone may help identifying these processes. We hypothesized that in small arteries accurate quantitation can be achieved during rhythmic vasomotion, and questioned whether these dynamics depend on intracellular signalling or physical vasoconstriction. We studied calcium-constriction dynamics in cannulated and pressurized rat mesenteric small arteries (,300 ,m in diameter). Combined application of tetra-ethyl ammonium (TEA) and BayK8644 induced rhythmicity, consisting of regular and irregular calcium spiking and superposition of spikes. Calcium spikes induced delayed vasomotion cycles. Their dynamic relation could be fitted by a linear second-order model. The dirac impulse response of this model had an amplitude that was strongly reduced with increasing perfusion pressure between 17 and 98 mmHg, while time to peak and relaxation time were the largest at an intermediate pressure (57 mmHg: respectively 0.9 and 2.3 sec). To address to what extent these dynamics reside in intracellular signalling or vasoconstriction, we applied rhythmic increases in pressure counteracting the vasoconstriction. This revealed that calcium-activation coupling became faster when vasoconstriction was counteracted. During such compensation, a calcium impulse response remained that lasted 0.5 sec to peak activation, followed by a 1.0 sec relaxation time, attributable to signalling dynamics. In conclusion, this study demonstrates the feasibility of quantitating calcium-activation dynamics in vasomoting small arteries. These dynamics relate to both intracellular sig-nalling and actual vasoconstriction. Performing such analyses during pharmacological intervention and in genetic models provides a tool for unravelling calcium-contraction coupling in small arteries. [source] Cholinergic modulation of angiogenesis: Role of the 7 nicotinic acetylcholine receptorJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2009Jenny C.F. Wu Abstract Pathological angiogenesis contributes to tobacco-related diseases such as malignancy, atherosclerosis and age-related macular degeneration. Nicotine acts on endothelial nicotinic acetylcholine receptors (nAChRs) to activate endothelial cells and to augment pathological angiogenesis. In the current study, we studied nAChR subunits involved in these actions. We detected mRNA for all mammalian nAChR subunits except ,2, ,4, ,, and , in four different types of ECs. Using siRNA methodology, we found that the ,7 nAChR plays a dominant role in nicotine-induced cell signaling (assessed by intracellular calcium and NO imaging, and studies of protein expression and phosphorylation), as well as nicotine-activated EC functions (proliferation, survival, migration, and tube formation). The ,9 and ,7 nAChRs have opposing effects on nicotine-induced cell proliferation and survival. Our studies reveal a critical role for the ,7 nAChR in mediating the effects of nicotine on the endothelium. Other subunits play a modulatory role. These findings may have therapeutic implications for diseases characterized by pathological angiogenesis. J. Cell. Biochem. 108: 433,446, 2009. © 2009 Wiley-Liss, Inc. [source] Lysophosphatidic acid in malignant ascites stimulates migration of human mesenchymal stem cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2008Mi Jeong Lee Abstract Lysophosphatidic acid (LPA) is elevated in ascites of ovarian cancer patients and is involved in growth and invasion of ovarian cancer cells. Accumulating evidence suggests a pivotal role of mesenchymal stem cells (MSCs) or stromal cells in tumorigenesis. In the present study, we demonstrated that ascites from ovarian cancer patients and LPA increased migration of human MSCs. The migration of MSCs induced by LPA and malignant ascites was completely abrogated by pretreatment with Ki16425, an antagonist of LPA receptors, and by silencing of endogenous LPA1, but not LPA2, with small interference RNA, suggesting a key role of LPA played in the malignant ascites-induced migration. LPA induced activation of ERK through pertussis toxin-sensitive manner, and pretreatment of MSCs with U0126, a MEK inhibitor, or pertussis toxin attenuated the LPA-induced migration. Moreover, LPA induced activation of RhoA in MSCs, and pretreatment of the cells with Y27632, a Rho kinase inhibitor, markedly inhibited the LPA-induced migration. In addition, LPA and malignant ascites increased intracellular concentration of calcium in MSCs, and Ki16425 completely inhibited the elevation of intracellular calcium. These results suggest that LPA is a crucial component of the malignant ascites which induce the migration of MSCs and elevation of intracellular calcium. J. Cell. Biochem. 104: 499,510, 2008. © 2007 Wiley-Liss, Inc. [source] Adapter protein CRKII signaling is involved in the rat pancreatic acini response to reactive oxygen speciesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2006Alberto G. Andreolotti Abstract Recent studies demonstrate that reactive oxygen species (ROS) are important mediators of acute pancreatitis, whether induced experimentally or in necrotizing pancreatitis in humans; however, the cellular processes involved remain unclear. Adapter protein CrkII, plays a central role for convergence of cellular signals from different stimuli. Cholecystokinin (CCK), which induces pancreatitis, stimulates CrkII tyrosine phosphorylation and CrkII protein complexes, raising the possibility it can be important in the acinar cell responses to ROS. Therefore, our aim was to investigate whether CrkII signaling is involved in the biological response of rat pancreatic acini to H2O2 and the intracellular mediators implicated. Treatment of isolated rat pancreatic acini with H2O2 rapidly stimulates CrkII phosphorylation, measured as electrophoretic mobility shift and by using a phosphospecific antibody (pTyr221). Tyrosine kinase blocker B44 inhibits the higher phosphorylation state, demonstrating that it occurs mainly in tyrosine residues. H2O2 -induced CrkII phosphorylation is time- and concentration-dependent, showing maximal effect with 3 mM H2O2 at 5 min. The intracellular pathways induced by H2O2 leading to CrkII tyrosine phosphorylation do not involve PKC, intracellular calcium, PI3-K or the actin cytoskeleton integrity. ROS generation clearly promotes the formation of protein complex CrkII,PYK2. In conclusion, ROS clearly affect the key adapter protein CrkII signaling by two ways: stimulation of CkII phosphorylation and a functional consequence: formation of CrkII,protein complexes. Because of its central role in activating more distal pathways, CrkII might likely play an important role in the ability of ROS to induce pancreatic cellular injury and pancreatitis. J. Cell. Biochem. © 2005 Wiley-Liss, Inc. [source] | |||||||||||||||||||||||||||||||