Activation of acetylcholine synthesis in cat sympathetic ganglia: dependence on external choline and sodium-pump rate

Abstract

Acetylcholine synthesis in the perfused cat superior cervical ganglion is maximally activated without activation of release during a 10 min recovery in Locke solution following a 15 min period of Na-pump inhibition by perfusion with K-free Locke solution; choline (5 X 10(-5) M) being present throughout. This procedure combined with the use of very high rates of perfusion flow has now permitted an examination of the roles of choline uptake and Na in the activation of synthesis. The data were analysed by analysis of variance as a basis for assessing experimental error and by Bartlett's test to assess equality of variance. Significance of differences between groups was estimated from this analysis (see Appendix). By selective omission of choline, either with or without addition of hemicholinium-3 (HC-3), in the K-free or in the recovery period it was found that choline is only taken up for formation of acetylcholine in the recovery period. With the use of different concentrations of choline in the recovery period, and omission of choline in the K-free period, it was found that the rate of acetylcholine synthesis increased with increasing choline concentration in conformity with Michaelis-Menten kinetics. The choline concentration giving half-maximal synthesis rate was 3.6 microM. Addition of 10(-6) M-HC-3 during recovery completely abolished synthesis in the presence of 5 X 10(-6) M-choline, and 2.5 X 10(-7) M-HC-3 reduced it by 68%. These values for choline dependence and inhibitory potency of HC-3 are similar to those found for high affinity choline transport in brain synaptosomes, indicating that the same system operates in brain and in ganglia. In additional experiments in which choline was omitted in the K-free period and with 5 X 10(-5) M-choline in the recovery fluid a reduction of external Na to 50 mM during recovery did not reduce significantly the maximal rate of acetylcholine synthesis. Further reduction to 25 nM, which would be expected to abolish the Na gradient, reduced the rate of synthesis by only 18%. The presence of 2 X 10(-5) M-ouabain during recovery in normal Locke solution containing 5 X 10(-5) M-choline abolished synthesis. It is concluded that choline uptake for acetylcholine synthesis in ganglia is via the high affinity transporter; that the transport is rate limiting for acetylcholine synthesis and; that the transport process is intimately linked to Na-pump rate.(ABSTRACT TRUNCATED AT 400 WORDS)