N-methyl-D-aspartate evokes rapid net depolymerization of filamentous actin in cultured rat cerebellar granule cells.
Filamentous actin (F-actin) was measured in cultured rat cerebellum granule neurons with the use of fluorescently labeled phallotoxin as a site-specific probe for F-actin, and fluorescence microscopy. The averaged apparent intensity of soma-associated F-actin-derived fluorescence (F(app)) was measured from fixed cells after incubation in either 1) normal Krebs solution containing 2 mM extracellular calcium ([Ca2+]ex) or 2) normal Krebs solution plus N-methyl-D-aspartate (NMDA) for 2 min immediately before fixation. NMDA (10, 50, and 100 microM) decreased F(app) to 63 +/- 5% (mean +/- SE), 53 +/- 4%, and 47 +/- 2%, respectively, of that measured from control cells. This effect was mimicked by treatment of cells with ionomycin. The ability of NMDA to reduce the F(app) in the presence of [Ca2+]ex was abolished when cells were maintained in [Ca2+]ex-free medium. Cells first treated with NMDA for 2 min and then left in normal medium for 30 min before fixation gave F(app) fluorescence similar to control values (91 +/- 12%). However, if the F-actin polymerization inhibitor cytochalasin D was added to cells immediately after NMDA was removed, the F(app) did not recover with time (36 +/- 3%). Cells treated for 30 min with cytochalasin D alone showed a small reduction in staining (approximately 20%). It is concluded that the actin polymerization state of rat cerebellar granule neurons is sensitive to changes in intracellular calcium, and that NMDA receptor activation evokes an initial rapid depolymerization of F-actin.
Shorte
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SL
Institut National de la Santé et de la Recherche Médicale (INSERM), Unité 29, Laboratoire de Neurobiologie et Physiopathologie du Développement, Hôpital de Port-Royal, Paris, France.
D000199:Actins / Q000187:drug effects* / Q000378:metabolism; D000546:Amanitins; D000818:Animals; D001704:Biopolymers; D002118:Calcium / Q000494:pharmacology; D048429:Cell Size / Q000187:drug effects; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D015638:Cytochalasin D / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D016202:N-Methylaspartate / Q000494:pharmacology*; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013268:Stimulation, Chemical; D013997:Time Factors
D000199:Actins; D000546:Amanitins; D001704:Biopolymers; D015638:Cytochalasin D; C018358:phallotoxin; D016202:N-Methylaspartate; D002118:Calcium