The experiments reported here were carried out to determine the effect of triethylcholine on synthesis of acetylcholine by sub-cellular fractions of rabbit brain homogenates. The choline acetylase (acetyl-CoA: eholine-O-acetyl-transferase, EC 2.3.1.6) incubation system used was that described by Hebb, 19634; acetyl-CoA is produced from acetylphosphate and coenzyme A, by phosphotrans-acetylase. The ACh synthesized was assayed biologically using the frog rectus abdominis muscle. Triethylcholine has been shown to be inactive on this assay preparation1 and it was confirmed that triethylcholine does not affect contractions due to acetylcholine.Table 1 shows the effect of triethylcholine on the synthesis of acetylcholine by the large granule or mitochondrial fraction of rabbit brain (P2 fraction) (ref. 4). When the concentration of choline (0.8 mg/tube) was optimal an equimolar concentration of triethylcholine (1.04 mg/tube) inhibited synthesis of acetylcholine by 11 per cent; doubling the triethylcholine concentration almost doubled the inhibition, and at the largest concentration of triethylcholine (10.4 mg/tube) the inhibition was 40 per cent. Table 1. INHIBITING ACTION OF TRIETHYLCHOLINB (TEC) ON SYNTHESIS OF ACETYLCHOLINE BY MITOCHONDRIA!, FRACTION OF RABBIT BRAINCholine Cl Added to the system Acetylcholine synthesized ^g/g P2 fraction % inh TEC 1-04 mg ibition produ TEC 2-08 mg iced by TEC 10-4 mg 0-2 mg 0-8 mg 3-2 mg 103 195 175 21 11 2-5 32 20 4-5 53 40 3-0Acet: plcholine synthesized P2 fraction (/"g/g) Choline Cl added No ether treatment 0-4 mg Ether treatment 0-4 mg Activity No TEC 108 ^g 438 ne TEC (4-16 mg) % 69 ftg 377 fig inhibition 36 140.8 mg choline Cl equimolar with 1.04 mg TEC Cl. Final volume of system on incubation = 0.1 ml. enzyme + 0.5 ml. Pre-incubation mixture = 0.6 ml. At a reduced concentration of choline (0.2 mg/tube) the same amounts of triethylcholine produced a greater inhibition of synthesis of acetylcholine. However, when the concentration of choline was increased 4-fold, triethylcholine had no significant effect on enzymatic synthesis of acetylcholine.Treatment with ether is known to activate choline acetylase in brain homogenates5, possibly by breakdown of membranes enclosing the enzyme. Comparisons have been made of the effect of triethylcholine on synthesis of acetylcholine by P2 fractions before and after treating them with ether, which caused a 4-fold increase in the activity of the fraction (Table 2). The results show that while triethylcholine still inhibited enzymatic synthesis of acetylcholine the effect, expressed as a percentage, was much smaller than that obtained with untreated particulates. Table 2. INHIBITING ACTION OF TRIETHYLCHOLINE ON SYNTHESIS OF ACETYLCHOLINE BY ETHER AND NON-ETHER TREATED MITOCHONDRIAL FRACTIONS OF RABBIT BRAINAcetylcholine synthesized (/wg/g) P2 fraction Choline Cl added No TECNo ether treatment 0-4 mg 108 ,ugEther treatment 0-4 mg 438 pgActivity Ether treated : non-ether treated fraction -438 : 108, approx. 4:1.Similar experiments using tissue slices obtained from rabbit caudate nucleus also demonstrated the inhibition of synthesis of acetylcholine by triethylcholine in the presence of low choline concentrations. These results indicate that triethylcholine inhibits synthesis of acetylcholine more readily when the tissue binding of the choline acetylase is not disturbed and that the inhibiting action of triethylcholine can be antagonized by using higher levels of choline. This suggests that triethylcholine is acting like hemicholinium by competing with choline for access to the enzyme through some membrane. It may also possess a slight direct inhibiting action on choline acetylase since in ether-treated particulates synthesis of acetylcholine was still impaired to some extent by large concentrations of triethylcholine, although here again the inhibition could be overcome by increasing the choline concentration.Taken as a whole the results described here provide an explanation of the effects of triethylcholine in vivo and support the hypothesis advanced by Bowman and Rand that this is due to impairment of synthesis of acetylcholine. One of us (B. A. H.) holds a Medical Research Council scholarship. We thank Ward, Blenkinsop and Co., Ltd., for a gift of triethylcholine chloride.