2. Exchangeability of radioactive acetylcholine with the bound acetylcholine of synaptosomes and synaptic vesicles
R M Marchbanks Biochem J. 1968 Jan;106(1):87-95. doi: 10.1042/bj1060087.
1. The exchangeability with added radioactive acetylcholine of the acetylcholine in isolated presynaptic nerve terminals (synaptosomes) and isolated synaptic vesicles was studied by a Sephadex-column method. 2. A substantial proportion of the synaptosomal acetylcholine is exchangeable with added radioactive acetylcholine. It is liberated by hypo-osmotic shock and ultrasonic treatment, and behaves as though it occupies the cytoplasmic compartment of synaptosomes. 3. Methods of isolating vesicles from hypo-osmotically ruptured synaptosomes in optimum yield are discussed. 4. The acetylcholine of synaptic vesicles isolated on a sucrose density gradient is released by hypo-osmotic conditions, suggesting that it is enclosed by a semi-permeable membrane; however, it is not easily released by ultrasonic treatment. 5. Added radioactive acetylcholine does not exchange with vesicular acetylcholine under a variety of different conditions. These include addition of ATP and Mg(2+), and pre-loading of the synaptosome with radioactive acetylcholine before hypo-osmotic rupture. This failure to exchange is discussed in terms of the possible storage mechanism of vesicular acetylcholine.
3. Choline metabolism in the cerebral cortex of guinea pigs. Stable-bound acetylcholine
L A Barker, M J Dowdall, V P Whittaker Biochem J. 1972 Dec;130(4):1063-75. doi: 10.1042/bj1301063.
1. The turnover of synaptosomal (vesicular-cytoplasmic) and stable-bound (vesicular) acetylcholine isolated from cortical tissue was investigated after the administration, under local anaesthesia, of [N-Me-(3)H]choline into the lateral ventricles of guinea pigs. 2. Radioactive acetylcholine and choline present in acid extracts of subcellular fractions were separated by a combination of liquid and column ion-exchange and thin-layer chromatography. 3. The specific radioactivity and pattern of labelling of acetylcholine present in a fraction of monodisperse synaptic vesicles was found to be essentially the same as that of synaptosomal acetylcholine. 4. The specific radioactivity of stable-bound acetylcholine present in partially disrupted synaptosomes (fraction H) at short times (10-20min) after the injection of [N-Me-(3)H]choline was very variable and inversely related to the yield of acetylcholine in that fraction. 5. Evidence was found for the existence of two small, but highly labelled pools of acetylcholine, one which could be isolated in fraction H and the other which was lost when synaptosomes, after isolation by gradient centrifugation, were left at 0 degrees C or pelleted. 6. It is concluded that the results are best explained by metabolic differences among the nerve-ending compartments (thought to be vesicles) which contain stable-bound acetylcholine. Computer simulation of our experiments supports this possibility and suggests that the highly labelled pool in fraction H is present in vesicles close to the external membrane.
