title: The phosphorylation status of extracellular-regulated kinase 1/2 in astrocytes and neurons from rat hippocampus determines the thrombin-induced calcium release and ROS generation.
abstract: Challenge of protease-activated receptors induces cytosolic Ca(2+) concentration ([Ca(2+) ](c)) increase, mitogen-activated protein kinase activation and reactive oxygen species (ROS) formation with a bandwidth of responses in individual cells. We detected in this study in situ the thrombin-induced [Ca(2+) ](c) rise and ROS formation in dissociated hippocampal astrocytes and neurons in a mixed culture. In identified cells, single cell responses were correlated with extracellular-regulated kinase (ERK)1/2 phosphorylation level. On average, in astrocytes, thrombin induced a transient [Ca(2+) ](c) rise with concentration-dependent increase in amplitude and extrusion rate and high ERK1/2 phosphorylation level. Correlation analysis of [Ca(2+) ](c) response characteristics of single astrocytes reveals that astrocytes with nuclear phosphoERK1/2 localization have a smaller Ca(2+) amplitude and extrusion rate compared with cells with a cytosolic phosphoERK1/2 localization. In naive neurons, without thrombin challenge, variable ERK1/2 phosphorylation patterns are observed. ROS were detected by hydroethidine. Only in neurons with increased ERK1/2 phosphorylation level, we see sustained intracellular rise in fluorescence of the dye lasting over several minutes. ROS formation was abolished by pre-incubation with the NADPH oxidase inhibitor apocynin. Additionally, thrombin induced an immediate, transient hydroethidine fluorescence increase. This was interpreted as NADPH oxidase-mediated O(2) (•-) -release into the extracellular milieu, because it was decreased by pre-incubation with apocynin, and could be eluted by superfusion. In conclusion, the phosphorylation status of ERK1/2 determines the thrombin-dependent [Ca(2+) ](c) increase and ROS formation and, thus, influences the capacity of thrombin to regulate neuroprotection or neurodegeneration.
authors: Institut für Neurobiochemie, Medizinische Fakultät, Otto-von-Guericke-Universität Magdeburg, Magdeburg, Germany.
authors: Gregor
authors: G
authors: Zündorf
authors: Georg
authors: G
authors: Reiser
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D006490:Hemostatics / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D005904:Glial Fibrillary Acidic Protein; D006490:Hemostatics; D017382:Reactive Oxygen Species; D011189:Potassium Chloride; D048049:Extracellular Signal-Regulated MAP Kinases; D013917:Thrombin; D010751:Phosphopyruvate Hydratase; D002118:Calcium