Article ; Online: 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.
2011 Volume 119, Issue 6, Page(s) 1194–1204
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 ... ...
| 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. |
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| MeSH term(s) | Animals ; Animals, Newborn ; Astrocytes/drug effects ; Astrocytes/metabolism ; Calcium/metabolism ; Cells, Cultured ; Dose-Response Relationship, Drug ; Enzyme Inhibitors/pharmacology ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Glial Fibrillary Acidic Protein/metabolism ; Hemostatics/pharmacology ; Hippocampus/cytology ; Neurons/drug effects ; Neurons/metabolism ; Phosphopyruvate Hydratase/metabolism ; Phosphorylation/drug effects ; Potassium Chloride/pharmacology ; Rats ; Reactive Oxygen Species/metabolism ; Thrombin/pharmacology |
| Chemical Substances | Enzyme Inhibitors ; Glial Fibrillary Acidic Protein ; Hemostatics ; Reactive Oxygen Species ; Potassium Chloride (660YQ98I10) ; Extracellular Signal-Regulated MAP Kinases (EC 2.7.11.24) ; Thrombin (EC 3.4.21.5) ; Phosphopyruvate Hydratase (EC 4.2.1.11) ; Calcium (SY7Q814VUP) |
| Language | English |
| Publishing date | 2011-12 |
| Publishing country | England |
| Document type | Journal Article ; Research Support, Non-U.S. Gov't |
| ZDB-ID | 80158-6 |
| ISSN | 1471-4159 ; 0022-3042 ; 1474-1644 |
| ISSN (online) | 1471-4159 |
| ISSN | 0022-3042 ; 1474-1644 |
| DOI | 10.1111/j.1471-4159.2011.07527.x |
| Database | MEDical Literature Analysis and Retrieval System OnLINE |
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