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Human Astrocytoma Cells (human + astrocytoma_cell)
Selected AbstractsLysophosphatidic Acid Inhibits Ca2+ Signaling in Response to Epidermal Growth Factor Receptor Stimulation in Human Astrocytoma Cells by a Mechanism Involving Phospholipase C, and a G,i ProteinJOURNAL OF NEUROCHEMISTRY, Issue 4 2000Marita Hernández Abstract: The effect of the lysophospholipid mediators lysophosphatidic acid (LPA) and sphingosine 1-phosphate and the polypeptide growth factor epidermal growth factor (EGF) on the human astrocytoma cell line 1321N1 was assessed. These agonists produced a rapid and transient increase of the intracellular Ca2+ concentration. When LPA was perfused before addition of EGF, the EGF-dependent Ca2+ transient was abrogated, whereas this was not observed when EGF preceded LPA addition. This inhibitory effect was not found for other EGF-mediated responses, e.g., activation of the mitogen-activated protein kinase cascade and cell proliferation, thus pointing to the existence of cross-talk between LPA and EGF for only a branch of EGF-induced responses. As 1321N1 cells expressed mRNA encoding the LPA receptors endothelial differentiation gene (Edg)-2, Edg-4, and Edg-7 and as sphingosine 1-phosphate did not interfere with LPA signaling, Edg-2, Edg-4, and/or Edg-7 could be considered as the LPA receptors mediating the aforementioned cross-talk. Attempts to address the biochemical mechanism involved in the cross-talk between the receptors were conducted by the immunoprecipitation approach using antibodies reacting with the EGF receptor (EGFR), phosphotyrosine, phospholipase C, (PLC,)-1, and G,i protein. LPA was found to induce coupling of PLC,-1 to the EGFR by a mechanism involving a G,i protein, in the absence of tyrosine phosphorylation of both PLC, and the EGFR. These data show a cross-talk between LPA and EGF limited to a branch of EGFR-mediated signaling, which may be explained by a LPA-induced, G,i -protein-mediated translocation of PLC,-1 to EGFR in the absence of detectable tyrosine phosphorylation of both proteins. [source] Interplay of constitutively released nucleotides, nucleotide metabolism, and activity of P2Y receptorsDRUG DEVELOPMENT RESEARCH, Issue 2-3 2001Eduardo R. Lazarowski Abstract At least six mammalian P2Y receptors exist that are specifically activated by ATP, UTP, ADP or UDP. Although the existence of ectoenzymes that rapidly metabolize extracellular nucleotides is well established, the relative flux of ATP and UTP through their extracellular metabolic products remains undefined. In addition, the existence of basal nucleotide release and the contribution of resting levels of ATP and UTP to P2 receptor activation are poorly understood. In the absence of exogenous agonists, an apyrase-sensitive inositol phosphate accumulation was observed in resting 16HBE14o, human bronchial epithelial cells endogenously expressing P2Y receptors and in 1321N1 human astrocytoma cells expressing a recombinant P2Y2 receptor. To test whether nucleotide release may account for basal P2 receptor activities, the rates of extracellular accumulation and metabolism of endogenous ATP were examined with resting 16HBE14o,, C6 rat glioma, and 1321N1 cell cultures. Although extracellular ATP concentrations (1-5 nM) remained unchanged for up to 12 h, [,32P] ATP included in the medium (as a radiotracer) was completely degraded within 120 min, indicating that ATP release balanced ATP hydrolysis. The calculated basal rates of ATP release ranged from 20 to 200 fmol/min per million cells. HPLC analysis during steady state revealed that the gamma-phosphate of ATP was reversibly transferred to species further identified as UTP and GTP, implicating ecto-nucleoside diphosphokinase (NDPK)-catalyzed phosphorylation of endogenous UDP and GDP. At steady state, the final 32P-products of [,32P]ATP metabolism were 32P-orthophosphoric acid and a species further purified and identified as 32P-pyrophosphate. Constitutive nucleotide release balanced by the concerted activities of ecto-ATPase, ecto-ATP pyrophosphatase, and ecto-NDPK may determine the resting levels of extracellular nucleotides and therefore, the basal activity of P2 receptors. Drug Dev. Res. 53:66,71, 2001. © 2001 Wiley-Liss, Inc. [source] The functional properties of the human ether-à-go-go -like (HELK2) K+ channelEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2002Andrea Becchetti Abstract The voltage-dependent K+ channels belonging to the ether-à-go-go family (eag, erg, elk) are widely expressed in the mammalian CNS. Their neuronal function, however, is poorly understood. Among the elk clones, elk2 is the most abundantly expressed in the brain. We have characterized the human ELK2 channel (HELK2) expressed in mammalian cell lines. Moreover, we have detected helk2 mRNA and ELK2-like currents in freshly dissociated human astrocytoma cells. HELK2 was inhibited by Cs+ in a voltage-dependent way (Kd was 0.7 mm, at ,120 mV). It was not affected by Way 123398 (5 µm), dofetilide (10 µm), quinidine (10 µm), verapamil (20 µm), haloperidol (2 µm), astemizole (1 µm), terfenadine (1 µm) and hydroxyzine (30 µm), compounds known to inhibit the biophysically related HERG channel. The crossover of the activation and inactivation curves produced a steady state ,window' current with a peak around ,20 mV and considerably broader than it usually is in voltage-dependent channels, including HERG. Similar features were observed in the ELK2 clone from rat, in the same experimental conditions. Thus, ELK2 channels are active within a wide range of membrane potentials, both sub- and suprathreshold. Moreover, the kinetics of channel deactivation and removal of inactivation was about one order of magnitude quicker in HELK2, compared to HERG. Overall, these properties suggest that ELK2 channels are very effective at dampening the neuronal excitability, but less so at producing adaptation of action potential firing frequency. In addition, we suggest experimental ways to recognize HELK2 currents in vivo and raise the issue of the possible function of these channels in astrocytoma. [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] | ||||||||||