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Ca2+ Chelator (ca2+ + chelator)
Selected AbstractsStimulation of fibroblast proliferation by neokyotorphin requires Ca2+ influx and activation of PKA, CaMK II and MAPK/ERKFEBS JOURNAL, Issue 2 2007Olga V. Sazonova Neokyotorphin [TSKYR, hemoglobin ,-chain fragment (137,141)] has previously been shown to enhance fibroblast proliferation, its effect depending on cell density and serum level. Here we show the dependence of the effect of neokyotorphin on cell type and its correlation with the effect of protein kinase A (PKA) activator 8-Br-cAMP, but not the PKC activator 4,-phorbol 12-myristate, 13-acetate (PMA). In L929 fibroblasts, the proliferative effect of neokyotorphin was suppressed by the Ca2+L -type channel inhibitors verapamil or nifedipine, the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane- N,N,N,,N, - tetraacetic acid acetoxymethyl ester, kinase inhibitors H-89 (PKA), KN-62 (Ca2+/calmodulin-dependent kinase II) and PD98059 (mitogen-activated protein kinase). The proliferative effect of 8-Br-cAMP was also suppressed by KN-62 and PD98059. PKC suppression (downregulation with PMA or inhibition with bisindolylmaleimide XI) did not affect neokyotorphin action. The results obtained point to a cAMP-like action for neokyotorphin. [source] Characterization of depolarization and repolarization phases of mitochondrial membrane potential fluctuations induced by tetramethylrhodamine methyl ester photoactivationFEBS JOURNAL, Issue 7 2005Angela M. Falchi Depolarization and repolarization phases (D and R phases, respectively) of mitochondrial potential fluctuations induced by photoactivation of the fluorescent probe tetramethylrhodamine methyl ester (TMRM) were analyzed separately and investigated using specific inhibitors and substrates. The frequency of R phases was significantly inhibited by oligomycin and aurovertin (mitochondrial ATP synthase inhibitors), rotenone (mitochondrial complex I inhibitor) and iodoacetic acid (inhibitor of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase). Succinic acid (mitochondrial complex II substrate, given in the permeable form of dimethyl ester) abolished the rotenone-induced inhibition of R phases. Taken together, these findings indicate that the activity of both respiratory chain and ATP synthase were required for the recovery of the mitochondrial potential. The frequency of D phases prevailed over that of R phases in all experimental conditions, resulting in a progressive depolarization of mitochondria accompanied by NAD(P)H oxidation and Ca2+ influx. D phases were not blocked by cyclosporin A (inhibitor of the permeability transition pore) or o -phenyl-EGTA (a Ca2+ chelator), suggesting that the permeability transition pore was not involved in mitochondrial potential fluctuations. [source] Chlorotoxin-sensitive Ca2+ -activated Cl, channel in type R2 reactive astrocytes from adult rat brainGLIA, Issue 4 2003Stanislava Dalton Abstract Astrocytes express four types of Cl, or anion channels, but Ca2+ -activated Cl, (ClCa) channels have not been described. We studied Cl, channels in a morphologically distinct subpopulation (, 5% of cells) of small (10,12 ,m, 11.8 ± 0.6 pF), phase-dark, GFAP-positive native reactive astrocytes (NRAs) freshly isolated from injured adult rat brains. Their resting potential, ,57.1 ± 4.0 mV, polarized to ,72.7 ± 4.5 mV with BAPTA-AM, an intracellular Ca2+ chelator, and depolarized to ,30.7 ± 6.1 mV with thapsigargin, which mobilizes Ca2+ from intracellular stores. With nystatin-perforated patch clamp, thapsigargin activated a current that reversed near the Cl, reversal potential, which was blocked by Cl, channel blockers, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and Zn2+, by I, (10 mM), and by chlorotoxin (EC50 = 47 nM). With conventional whole-cell clamp, NPPB- and Zn2+ -sensitive currents became larger with increasing [Ca2+]i (10, 150, 300 nM). Single-channel recordings of inside-out patches confirmed Ca2+ sensitivity of the channel and showed open-state conductances of 40, 80, 130, and 180 pS, and outside-out patches confirmed sensitivity to chlorotoxin. In primary culture, small phase-dark NRAs developed into small GFAP-positive bipolar cells with chlorotoxin-sensitive ClCa channels. Imaging with biotinylated chlorotoxin confirmed the presence of label in GFAP-positive cells from regions of brain injury, but not from uninjured brain. Chlorotoxin-tagged cells isolated by flow cytometry and cultured up to two passages exhibit positive labeling for GFAP and vimentin, but not for prolyl 4-hydroxylase (fibroblast), A2B5 (O2A progenitor), or OX-42 (microglia). Expression of a novel chlorotoxin-sensitive ClCa channel in a morphologically distinct subpopulation of NRAs distinguishes these cells as a new subtype of reactive astrocyte. GLIA 42:325,339, 2003. © 2003 Wiley-Liss, Inc. [source] Calcium in the mechanism of ammonia-induced astrocyte swellingJOURNAL OF NEUROCHEMISTRY, Issue 2009Arumugam R. Jayakumar Abstract Brain edema, due largely to astrocyte swelling, is an important clinical problem in patients with acute liver failure. While mechanisms underlying astrocyte swelling in this condition are not fully understood, ammonia and associated oxidative/nitrosative stress appear to be involved. Mechanisms responsible for the increase in reactive oxygen/nitrogen species (RONS) and their role in ammonia-induced astrocyte swelling, however, are poorly understood. Recent studies have demonstrated a transient increase in intracellular Ca2+ in cultured astrocytes exposed to ammonia. As Ca2+ is a known inducer of RONS, we investigated potential mechanisms by which Ca2+ may be responsible for the production of RONS and cell swelling in cultured astrocytes after treatment with ammonia. Exposure of cultured astrocytes to ammonia (5 mM) increased the formation of free radicals, including nitric oxide, and such increase was significantly diminished by treatment with the Ca2+ chelator 1,2-bis-(o -aminophenoxy)-ethane- N,N,- N,,N, -tetraacetic acid tetraacetoxy-methyl ester (BAPTA). We then examined the activity of Ca2+ -dependent enzymes that are known to generate RONS and found that ammonia significantly increased the activities of NADPH oxidase (NOX), constitutive nitric oxide synthase (cNOS), and phospholipase A2 (PLA2) and such increases in activity were significantly diminished by BAPTA. Pre-treatment of cultures with 7-nitroindazole, apocyanin, and quinacrine, respective inhibitors of cNOS, NOX, and PLA2, all significantly diminished RONS production. Additionally, treatment of cultures with BAPTA or with inhibitors of cNOS, NOX, and PLA2 reduced ammonia-induced astrocyte swelling. These studies suggest that the ammonia-induced rise in intracellular Ca2+ activates free radical producing enzymes that ultimately contribute to the mechanism of astrocyte swelling. [source] [Na+]i -induced c-Fos expression is not mediated by activation of the 5,-promoter containing known transcriptional elementsFEBS JOURNAL, Issue 14 2007Mounsif Haloui In vascular smooth muscle cells and several other cell types, inhibition of Na+/K+ -ATPase leads to the expression of early response genes, including c-Fos. We designed this study to examine whether or not a putative Na+i/K+i -sensitive element is located within the c-Fos 5,-UTR from ,,650 to +,103 containing all known response elements activated by ,classic' stimuli, such as growth factors and Ca2+i -raising compounds. In HeLa cells, the highest increment of c-Fos mRNA content was noted after 6 h of Na+/K+ -ATPase inhibition with ouabain that was abolished by actinomycin D, an inhibitor of RNA synthesis. c-Fos protein accumulation in ouabain-treated cells correlated with a gain of Na+i and loss of K+i. Augmented c-Fos expression was also observed under inhibition of Na+/K+ -ATPase in K+ -free medium and in the presence of the Na+ ionophore monensin. The effect of ouabain on c-Fos expression was sharply attenuated under dissipation of the transmembrane Na+ gradient, but was preserved in the presence of Ca2+ chelators and the extracellular regulated kinase inhibitor PD98059, thus indicating an Na+i -mediated, Ca2+i - and extracellular regulated kinase-independent mechanism of gene expression. In contrast to massive c-Fos expression, we failed to detect any effect of ouabain on accumulation of luciferase driven by the c-Fos 5,-UTR. Negative results were also obtained in ouabain-treated vascular smooth muscle cells and C11 Madin,Darby canine kidney cells possessing augmented c-Fos expression. Our results reveal that Na+i -induced c-Fos expression is not mediated by the 5,-UTR containing transcriptional elements activated by growth factors and other ,classic stimuli'. 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