Exocytotic Release of [3H]-Acetylcholine by Ouabain Involves Intracellular Ca2+ Stores in Rat Brain Cortical Slices

1. The effect of ouabain on the release of 3H]acetylcholine (3H]ACh) in rat brain cortical slices was investigated. 2. The ouabain-induced release of 3H]ACh was calcium-independent and not blocked by EGTA. 3. BAPTA-AM, a chelator of intracellular calcium, inhibited the ouabain effect suggesting the involvement of intracellular calcium stores. 4. Vesamicol, a drug that blocks the storage of acetylcholine in synaptic vesicles inhibited by 73% the ouabain-induced release of 3H] ACh, suggesting exocytotic release of the neurotransmitter. 5. Dantrolene and tetracaine, inhibitors of ryanodine and InP3 receptors, inhibited by 57 and 66% respectively, the ouabain-elicited release of 3H]ACh in brain cortical slices. 6. Confocal microscopy and calcium imaging showed that ouabain increased the levels of Ca2+]i in cholinergic SN56 cells and that this increase was concentrated in the cell soma. 7. In conclusion, we suggested that ouabain causes Ca2+ release from intracellular stores that can increase 3H] ACh exocytosis from rat brain cortical slices.