Characteristics of thrombin-induced calcium signals in rat astrocytes.
The protease thrombin seems to play a central role in events following neural injury, whereby the enzyme can act, in concert with other molecules as a hormone or as a growth factor. In cells derived from the nervous system, thrombin induces changes in morphology and proliferation. The signalling mechanisms involved in these thrombin-activated processes are still unclear. In the present study we investigated Ca2+ signals in fura-2 loaded rat astrocytes in primary culture. Brief stimulation of astrocytes with thrombin induced a dose-dependent transient elevation of [Ca2+]i, best fitted by a double-sigmoidal curve giving two EC50 values of 3 pM and 150 pM. Continuous superfusion of cells with thrombin induced Ca2+ responses with three different types of kinetics. In 48% of the cells tested a single transient rise superimposed with fast fluctuations of [Ca2+]i was seen. The following complex long-term changes of [Ca2+]i, dependent on the presence of the agonist thrombin, were observed: i) a biphasic [Ca2+]i elevation, characterized by an initial peak followed by a sustained plateau phase (in 43% of the cells) and ii) oscillations of [Ca2+]i (in 9% of the cells). The observed Ca2+ responses were inhibited by the phospholipase C (PLC) inhibitor U-73122 and the thrombin inhibitor protease nexin-1/glia-derived nexin. The synthetic thrombin receptor activating peptide could mimic the thrombin-induced changes of [Ca2+]i. In astrocytes in Ca2+-free medium, thrombin induced a sharp single transient Ca2+ rise, without superimposed fluctuations. After depletion of intracellular Ca2+ stores with thapsigargin the Ca2+ response to thrombin was diminished or completely suppressed indicating that thrombin induces the release of Ca2+ from intracellular stores. During long-term Ca2+ responses, omission of extracellular Ca2+ resulted in a reversible interruption of the signal. In conclusion our results demonstrate that thrombin by activation of its plasma membrane receptor induces through activation of PLC different types of Ca2+ responses. The complex Ca2+ signals are generated by an interplay of InsP3-mediated Ca2+ release from intracellular stores and Ca2+ entry across the plasma membrane.
Ubl
J J
JJ
Institut für Neurobiochemie, Medizinische Fakultät, Otto-von-Guericke-Universität Magdeburg, Germany.
Reiser
G
G
D016564:Amyloid beta-Protein Precursor; D000818:Animals; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism; D002118:Calcium / Q000502:physiology*; D002352:Carrier Proteins / Q000494:pharmacology; D002478:Cells, Cultured; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D015849:Plasminogen Inactivators / Q000494:pharmacology; D058255:Protease Nexins; D051381:Rats; D011956:Receptors, Cell Surface; D015398:Signal Transduction / Q000187:drug effects*; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors
D016564:Amyloid beta-Protein Precursor; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D005904:Glial Fibrillary Acidic Protein; D015849:Plasminogen Inactivators; D058255:Protease Nexins; D011956:Receptors, Cell Surface; D010738:Type C Phospholipases; D013917:Thrombin; D002118:Calcium