title: Modulation of ion gradients and glutamate release in cultured cerebellar granule cells by ouabain.
abstract: Upon addition of the cardiac glycoside ouabain to cultured cerebellar granule cells, an immediate increase in intracellular free sodium is evoked mediated by two pathways, a voltage-sensitive channel blocked by tetrodotoxin and a channel sensitive to flunarizine. Ouabain induces a steady plasma membrane depolarization in low Ca2+ medium; whereas in the presence of Ca2+, a distinct discontinuity is observed always preceded by a large increase in intracellular free Ca2+ ([Ca2+]c). The plateau component of the increase can be inhibited additively by the L-type Ca2+ channel antagonist nifedipine, the spider toxin Aga-Gl, and the NMDA receptor antagonist MK-801. Single-cell imaging reveals that the [Ca2+]c increase occurs asynchronously in the cell population and is not dependent on a critical level of extracellular glutamate or synaptic transmission between the cells. A prolonged release of glutamate is also observed that is predominantly Ca2+ dependent for the first 6-10 min after the evoked increase in [Ca2+]c. This release is four times as large as that observed with 50 mM KCl and is predominantly exocytotic because release was inhibited by tetanus toxin, the V-type ATPase inhibitor bafilomycin, and Aga-Gl. It is proposed, therefore, that ouabain induces a period of membrane excitability culminating in a sustained exocytosis above that observed upon permanent depolarization with KCl.
authors.affiliation: Department of Biochemistry, Medical Sciences Institute, University of Dundee, Scotland.
authors.forename: M A
authors.initials: MA
authors.lastname: Cousin
authors.forename: D G
authors.initials: DG
authors.lastname: Nicholls
authors.forename: J M
authors.initials: JM
authors.lastname: Pocock
mesh_terms: D018750:6-Cyano-7-nitroquinoxaline-2,3-dione / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002462:Cell Membrane / Q000502:physiology; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016291:Dizocilpine Maleate / Q000494:pharmacology; D018698:Glutamic Acid / Q000378:metabolism*; D007473:Ion Channels / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009543:Nifedipine / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D012964:Sodium / Q000378:metabolism; D013111:Spider Venoms / Q000494:pharmacology
chemical_list: C077471:Aga-GI toxin; D007473:Ion Channels; D013111:Spider Venoms; D018698:Glutamic Acid; D010042:Ouabain; D016291:Dizocilpine Maleate; D018750:6-Cyano-7-nitroquinoxaline-2,3-dione; D012964:Sodium; D009543:Nifedipine; D002118:Calcium