Is any Free Acetylcholine Preformed in Resting Muscles or in the Hertrt?1

The presence of free acetylcholine in extracts from various organs has been secured by many investigators using chemical as well as biological methods for the identification. The discussion of pharmacological evidences allowed the conclusion, however, that this free acetylcholine found in the extracts is not preformed in resting organs. The discussion also supported the working hypothesis that the free acetylcholine in the extracts is formed through breakdown of a preformed complex. It was assumed that the breakdown of this complex under liberation of free acetylcholine is favoured by the methods hitherto used for the determination of acetylcholine in tissues. The experiments presented in this communication have given the following results: Resting organs were frozen with liquid air and extracted at a low temperature with ethyl alcohol. The alcohol was evaporated and the extract concentrated in vacuo. This extract was free from cells and from proteins. It contained no free acetylcholine, although added acetylcholine was quantitatively refound. If the extract was acidified and heated, an active substance was formed, which was found to be acetylcholine. The same results were obtained with trichloracetic acid extracts if only the organs to be extracted were frozen with liquid, air. the extraction made at a low temperature and if the ex of trichloracetic acid was removed as soon as possible. The formation of acetylcholine at the heating of acidified extracts is not due to a synthesis. The extracts are free from cells and as far as known free from proteins; addition of choline and acetic acid has no influence on the formation of acetylcholine: the formation can also be shown in extracts where all free choline has been removed through extraction with acetone. The formation of acetylcholine is due to breakdown of a labile complex compound, which we call “precursor” as we know nothing about its chemical constitution. The precursor was found to have the following properties: Like free acetylcholine it is soluble in water, in ahsolute alcohol and in mixtures of water and alcohol, and insoluble in ether and petrol ether. Unlike free acetylcholine and choline it is insoluble in acetone and can be precipitated with acetone from a watery solution. It is precipitated with bariuni acetate from neutral or slightly acid watery or alcoholic solutions. It is relatively stable at pH 4. At other acidities it breaks down liberating free acetylcholine. This process is considerably more rapid than the hydrolysis of free acetylcholine. The free acetylcholine earlier found in extracts from resting organs is formed through breakdown of the precursor during the analysis. The experiments allow the following conclusion: Resting organs contain no preformed acetylcholine. Tissue acetylcholine is present as an integral part of a labile complex compound, which differs from free acetylcholine through its chemical properties.