The Effects of Botulinum Neurotoxin and Tetrodotoxin on Protein Phosphorylation in Pure Cholinergic Synaptosomes
Protein phosphorylation has been related to the molecular mechanisms of neurotransmitter release (1) and some phosphoproteins as Synapsin I are thought to be implicated in the exocytotic process (2). On the other hand, depolarization of isolated synaptic buttons (synaptosomes) results in the phosphorylation of specific proteins in rat brain synaptosomes (3) and in Torpedo electric organ synaptosomes (4). The electric organ of Torpedo is a useful model to study the acetylcholine release since it is innervated exclusively by cholinergic nerves (5), and presents an homologous structure with the neuromuscular junction. In contrast, in the electric organ of Torpedo the presynaptic nerve terminals represent as much as 2% to 3% of the total volume. A pure cholinergic synaptosomal fraction can be isolated from this organ (6). In these cholinergic synaptosomes the induced release of acetylcholine (7) and ATP (8) and the calcium fluxes (9) have been characterized. Acetylcholine release can be induced by several chemical depolarising agents as high external potassium concentration, or veratridine or the calcium ionophore A 23187. Botulinum toxin (BoNTx) blocks the acetylcholine release (see 10 and 11 for reviews) in several preparations. In cholinergic synaptosomes from the electric organ of Torpedo marmorata BoNTx inhibits the induced acetylcholine release whereas ATP release, calcium uptake and membrane potential are not affected by the toxin (12). We have used this pure cholinergic preparation to study the effect of botulinum neurotoxin type A under depolarising conditions on the rate of phosphorylation of synaptosomal proteins.
KeywordsSucrose Phosphorus Urea Amide Electrophoresis
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