CHOLINERGIC STIMULATION OF PHOSPHOLIPID LABELLING FROM [32P]ORTHOPHOSPHATE IN GUINEA‐PIG CORTEX SYNAPTOSOMES IN VITRO: SUBSYNAPTOSOMAL LOCALIZATION

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Abstract
Abstract— Subsynaptosomal localization of stimulation of phospholipid labelling by cholinergic agents was investigated. Synaptosomes prepared from guinea‐pig cortex were incubated with [32P]orthophosphate in the presence or absence of 10−3m carbamylcholine. Following incubation and osmotic shock, lysed synaptosomes were subjected to density gradient fractionation. Subsynaptosomal fractions were examined by electron microscopy and analysed for enzyme activities and 32P‐labelled lipids.In the absence of carbamylcholine, labelled phosphatidate and phosphatidylinositol were recovered in layers and interfaces A, B, C and D formed over 0.9, 1.1, 1.2 and 1.3 m sucrose, with highest amounts of label in fractions C and D for both lipids. Carbamylcholine induced the greatest increment in these two labelled lipids in fractions A and B. This distribution correlated with the presence of acetylcholinesterase activity and membrane ghosts. No correlation was found among the four fractions between the induced increase in labelling and succinic dehydrogenase activity or with the abundance of mitochondria, synaptic vesicles, or cytoplasmic fragments identified by electron microscopy. In contrast with the increases seen in phosphatidylinositol and phosphatidate labelling, carbamylcholine caused a decrease in 32P‐labelling of the polyphosphoinositides, and this effect was seen primarily in the heavier subsynaptosomal fractions, C and D.

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