MODULATION OF ION GRADIENTS AND GLUTAMATE RELEASE IN CULTURED CEREBELLAR GRANULE CELLS BY OUABAIN

Upon addition of the cardiac glycoside ouabain to cultured cerebellar granule cells, an immediate increase in intracellular free sodium is e voked 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 pre ceded by a large increase in intracellular free Ca2+ ([Ca2+](i)). The plateau component of the increase can be inhibited additively by the L -type Ca2+ channel antagonist nifedipine, the spider toxin Aga-GI, and the NMDA receptor antagonist MK-801. Single-cell imaging reveals that the [Ca2+](c) increase occurs asynchronously in the cell population a nd is not dependent on a critical level of extracellular glutamate or synaptic transmission between the cells. A prolonged release of glutam ate is also observed that is predominantly Ca2+ dependent for the firs t 6-10 min after the evoked increase in [Ca2+](c). This release is fou r times as large as that observed with 50 mM KCI and is predominantly exocytotic because release was inhibited by tetanus toxin, the V-type ATPase inhibitor bafilomycin, and Aga-GI, It is proposed, therefore, t hat ouabain induces a period of membrane excitability culminating in a sustained exocytosis above that observed upon permanent depolarizatio n with KCI.