(14C) acetylcholine synthesis by cortex slices of rat brain.

Abstract

Abstract— A procedure has been developed to measure ACh synthesis from [14C]‐precursors. As little as 10−9 moles of ACh were detected as the result of de nova synthesis. Following incubation of cortex slices of rat brain with eserine and a tagged metabolite, ACh carrier was added to the incubation medium and to an extract from the slices. ACh was purified by chromatography on Amberlite CG‐50, precipitation and recrystallization of ACh chloroaurate. [U−14C]glucose and [2−14C]pyruvate formed similar amounts of [14C]ACh. Hydrolysis of ACh with subsequent chromatography of the resultant acetic acid demonstrated that all of the label was located in the acetyl moiety. [14C]acetate did not serve as a precursor of the acetyl group of ACh. Equivalent incorporation of carbons 1 and 6 of glucose into ACh indicated that glucose metabolism to ACh occurred via the Embden‐Meyerhof pathway. The amount of ACh detected by bioassay after incubation of cortex slices with [U−14C]glucose was approximately the same as that calculated as labelled ACh; this demonstrates that all of the acetyl groups of ACh formed during incubation were derived from glucose. [14C]choline, either methyl or chain labelled, formed [14C]ACh while labelled ethanolamine, serine and methionine did not. Synthesis from labelled choline did not occur in the absence of glucose. When both [U−14C]glucose and [14C]choline were incubated with brain slices, the acetyl and choline moieties of ACh were equally labelled; this demonstrates that the entire molecule was formed from added precursors. Slices supported a high rate of ACh synthesis without addition of choline. The addition of 10−4m‐hemicholinium‐3 inhibited ACh formation by more than 90 per cent from either [U‐14C]glucose or [Me‐14C]choline. Study of the time course of ACh synthesis from glucose demonstrated a rapid formation of [14C]ACh within the slices which reached a maximum during the first hour of incubation. [14C]ACh in the incubation medium accumulated at a linear rate for 3 hr. Replacement of a portion of the sodium chloride of the incubation medium by potassium chloride to a final concentration of 31 mm‐KCI markedly increased the formation of [14C]ACh found in the incubation medium. Decreased amounts of [14C]ACh were extracted from the slices by homogenization or by subsequent heating at pH 4 in the high potassium ion medium.