Bibliothek

Notizen: Chemiluminescent detection was applied to measure the continuous spontaneous Ca2+-independent liberation of acetylcholine (ACh) from Torpedo electric organ synaptosomes. Differentiation between the release of ACh and choline was achieved by inhibiting cholinesterases with phospholine, and a way to quantify the continuous release was devised. The method permitted measurements during short time intervals from minute amounts of tissue and without an accumulation of ACh in the medium. Synaptosomes continuously liberated small amounts of ACh during incubations in the presence of 3 mM K+ and in the absence of Ca2+. The spontaneous liberation of ACh was similar both quantitatively and qualitatively at pH values of 8.6 and 7.8. It was unaltered by MgCl2 (10.4 mM), 2-(4-phenylpiperidino)cyclohexanol (10 μM), ouabain (104 μM), atropine (10μM), and valinomycin (102 nM). Carbamoylcholine brought about a decrease, which could be partially reversed by atropine. The Ca2+-independent output of ACh was increased considerably when the concentration of K+ ions was raised (eightfold at 103 and 35-fold at 203 mM K+). Carbamoylcholine (104 μM) blocked the increase in ACh release produced by high K+; this effect of carbamoylcholine was not reversed by atropine (10 μM). When Ca2+ was added to synaptosomes depolarized by a high concentration of K+, the amount of ACh released during the first 1–3 min after the addition of Ca2+ was at least 20 times higher than in the absence of Ca2+, but the release returned rapidly to predepolarization values. Similarly high values of ACh release could be achieved by adding Ca2+ plus the ionophore A23187 and even higher values by adding Ca2+ plus gramicidin. Under the conditions used. the Ca2+-dependent K+-evoked release of ACh was not changed by 104 μM carbamoylcholine. It is apparent that the spontaneous Ca2+-independent liberation of ACh from Torpedo synaptosomes is not a simple diffusion and is mediated by a carrier. The carrier is inhibited by muscarinic activation; the molecular mechanism of the inhibition is unlikely to involve Ca2+ ions or hyperpolarization. During depolarization, muscarinic inhibition of ACh liberation is ineffective, but carbamoylcholine blocks the Ca2+-independent depolarization-stimulated ACh output, probably by a direct action on the carriers.