Neurochemistry of Cholinergic Terminals

SUMMARY1. Normal and denervated rat diaphragms and neural (central) and aneural (peripheral) parts of normal diaphragms were incubated under several different conditions likely to affect the metabolism of acetylcholine (ACh), with the aim of discovering specific features of the control of neural and aneural ACh in the muscle. The concentrations of ACh in the tissue and the medium were measured at the end of the incubations using a radioenzymatic assay, and the amount of ACh synthesized during the incubations was calculated by subtracting the initial amount of ACh present in the tissue from that found in the tissue plus the medium at the end of the incubations.2. Confirming earlier results obtained with bioassays, it was found that, in a medium with 5 mM-K+ and 2-5 mM-Ca2+, denervated diaphragms released ACh into the medium at a rate equal to 47 % of that observed in normal diaphragms; the amount of ACh released from aneural parts of normal diaphragms was 51 % of that released from their neural parts. The release from normal diaphragms was increased (83 %) in a Ca2+-dependent manner by raising the concentration of K+ to 30 mm. In the denervated diaphragms, 30 mM-K+ brought about a Ca2+-independent increase (67 %) in the rate of ACh release. The elevation of K+ was without effect on the release of ACh from aneural parts of normal diaphragms.3. The results indicate that a Ca2+-dependent mechanism of ACh release, known to function in the nerve terminals, is not likely to participate in the efflux of ACh from the muscle fibres. The K+-induced but Ca2+-independent enhancement of ACh release from the denervated diaphragms probably occurs by diffusion of ACh along the altered electrochemical gradient. It is suggested that the surface membranes of the muscle fibres become more permeable to ACh after denervation.4. During incubations with 30 mM-K+and 10 gM-hemicholinium-3 (HC-3), an inhibitor of the carrier-mediated transport of choline, the rates of ACh release and synthesis in normal diaphragms were diminished to the levels found in the denervated diaphragms, in which the concentration, release and synthesis of ACh were not affected by HC-3. The synthesis of aneural ACh thus appears to be independent of the carrier-mediated supply of choline across cell membranes.5. The release of ACh from normal diaphragms incubated with 5 mM-K+ was V. DOLEZAL AND S. TUCEK increased in the presence of 100 ,uM-ouabain, whereas the release from denervated diaphragms was not affected. This finding suggests that the mechanism of ACh release that is activated by ouabain in the nerve cells involves, in addition to the inhibition of Na+-K+-ATPase, some other steps which are not operative in the muscle fibres. 6. The results corroborate earlier evidence indicating that aneural ACh is produced, stored and released in the diaphragms. They fit the view that the aneural ACh is located in the cytoplasm of the muscle fibres and that it leaves the muscle fibres by molecular 'leakage' rather than by a specialized release mechanism. The efflux ofACh fr...

Section: The Content Of Ach In the Diaphragmsupporting

confidence: 81%

The synthesis and release of acetylcholine in normal and denervated rat diaphragms during incubation in vitro

Doležal

Tuček

1983

“…This was done by examining the potency of HC-3 in inhibiting activation of synthesis because the high-affinity choline transport system is considerably more sensitive to inhibition by HC-3 than is the low-affinity system. Published values for the K1 (the inhibition constant) of HC-3 in brain synaptosomes range from 0-025 to 1-0 ,UM for the high-affinity system and from 5 to 120 #UM for the low-affinity system (for references: MacIntosh & Collier, 1976;Martin, 1977).…”

Section: Methodsmentioning

confidence: 99%

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

Worsley¹,

Birks²

1985

SUMMARY1. 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.2. 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).3. 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.4. 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 /M.5. 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. 6. 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 mm, which would be expected to abolish the Na gradient, reduced the rate of synthesis by only 18 %.

“…This would be surprising since the uptake of intact ACh is usually considered as a much less effective process than that of choline or acetate (see MacIntosh & Collier, 1976).…”

Section: Discussionmentioning

confidence: 99%

Acetylcholine release evoked by single or a few nerve impulses in the electric organ of Torpedo.

Dunant¹,

Eder²,

Servetiadis-Hirt³

1980

SUMMARY1. The acetylcholine (ACh) store in the Torpedo electric organ was partially labelled with choline and acetate at the same molar concentration but with different isotopes. Under these conditions the two precursors were incorporated into ACh in a ratio 1 to 1.2. After a single electrical stimulus, or a brief burst of stimuli, the compound electroplaque potential (e.p.p.) was recorded and the radioactive choline and/or acetate counted in the perfusion fluid, providing a sensitive assay for ACh release in the absence of anticholinesterase drugs.3. The so-called depression of transmission was found to be due to progressive impairment of ACh release in the successive impulses evoked by repeated stimuli.4. In a pair of impulses separated by 50 ms interval, less ACh was released by the second than by the first impulse; this explained why the size of the second e.p.p. was depressed, using a direct measurement of ACh.5. In repetitive stimulations of longer duration, the maximum rate of release declined as the activity was prolonged. Thus the tissue progressively lost its ability to ensure release at high frequencies.6. An unexpected finding was that anticholinesterases like eserine or pre-treatment with fluostigmine (DFP) greatly reduced ACh release even by a single impulse.7. Evoked ACh release and e.p.p. amplitude were both maximum between 10 and 20 'C. At higher temperatures, the evoked release decreased as the spontaneous release increased.8. Changes in external Ca2+ and Mg2+ produced similar changes in the e.p.p. and evoked ACh release. The dose-response curve for Ca dependency of ACh release was very steep with a Hill's coefficient of 3 2.9. With a single stimulus in the presence of 4-aminopyridine, there was a dramatic enlargement of the e.p.p. and a still larger potentiation of the evoked ACh release. 10. It has been possible with this approach to avoid the inconveniences often encountered in similar studies, i.e. repetitive stimulation, low Ca solutions and cholinesterase inhibition. This permitted a good correlation between electrophysiological and biochemical estimates of transmitter release even by a single nerve impulse.