The Effect of Ouabain on the Release of [¹⁴C]Acetylcholine and Other Substances from Synaptosomes

Abstract

The effect of ouabain and dihydroouabain on Na+‐K+ ATPase, 86Rb uptake and the release of [14C]ACh (acetylcholine) from synaptosomal preparations of guinea pigs was compared. At low concentrations of glycoside (<50 μm) there was a good correlation between the potency of ouabain and of dihydroouabain in inhibiting Na+‐K+ ATPase and in causing the release of [l4C]ACh in a nondepolarising medium. Ouabain (200 μM) increased the release of [14C]ACh evoked by 25 mm‐KCl, but not that evoked by 100μm‐veratrine. The enhancement of release was independent of the presence of calcium. It was observed that in addition to [14C]ACh release, choline efflux was also stimulated by ouabain, independently of the presence of Ca2+. Experiments with hemicholinium‐3 showed that the ouabain‐induced increase in choline efflux was not due to an inhibition of reuptake. The effect of ouabain on intrasynaptosomal K+ concentration was measured in order to investigate the degree of depolarisation it caused. The decrease in K+ was found to be similar in magnitude and time course to that caused by veratrine. It was shown that ouabain‐induced depolarisation caused an increased efflux of another positive ion (dibenzyldimethylammonium chloride) and retention of a negatively charged ion (chloride), as would be expected from the operation of the electrochemical potential gradient changing as a result of depolarisation. It is suggested that ouabain acts to stimulate ACh release from synaptosomes as follows: following blockage of the Na+‐K+ ATPase there is rapid depolarisation which, if Ca2+ is present, provokes the normal Ca2+‐dependent transmitter release process to occur. In addition, depolarisation accelerates the leakage of positive ions down their electrochemical potential gradient, but causes a retention of negative ions. Such an action does not depend on the presence of Ca2+, nor is it specific to transmitters.

External Ca2+‐Independent Release of Neurotransmitters

“…Ouabain increases the intrasynaptosomal Na+ concentration by inhibiting the plasma membrane Na+, K+-ATPase (Archibald and White, 1974;Coutinho et al, 1984) and thus consequently causes depolarization (Adam- Vizi and Ligeti, 1984). It has been reported to induce release of ACh from the Auerbach plexus of the gut (Paton et al, 197 1;Vizi, 1977), brain slices (Vizi, 1972), and synaptosomes (Meyer and Cooper, 1981;Vyas and Marchbanks, 1981;AdamVizi and Ligeti, 1984). From the observation that it enhanced the spontaneous nonquantal ACh release from motor nerve terminals (Vyskotil and Illes, 1977;Vizi and Vyskoeil, 1979), an important conclusion could be drawn, namely, that a nonvesicular ACh pool is likely to be involved in the release.…”

Release of acetylcholine from rat brain synaptosomes by various agents in the absence of external calcium ions.

“…It has already been shown that ouabain (Vizi, 1972(Vizi, , 1977Baker & Crawford, 1975;Sandoval, 1980;Meyer & Cooper, 1981;Vyas & Marchbanks, 1981) veratridine (Meyer & Cooper, 1981, Schoffelmeer & Mulder, 1983 are also able to increase transmitter release from different tissues under these conditions. Our aim was to follow not only the change in ACh release but also 86Rb+ distribution.…”

“…Vyas & Marchbanks (1981) suggested that in the presence of extracellular Ca2+ a 'normal', depolarization-induced, Ca2+-dependent release process is produced in synaptosomes by ouabain whereas in the absence of external Ca2+ an unspecific change in membrane permeability independent of depolarization leads to increased transmitter release. Our finding, the clearly similar character of the relationship between membrane potential and ACh release in the presence and absence of external Ca2+, would suggest one principal mechanism operating in both cases.…”

“…All these agents are known to depolarize the membrane; however, the effect of a prolonged depolarization either by chemical agents or by repetitive stimulation seems to differ in some respects from that ofa briefnerve impulse. Tetanic stimulation increases the frequency ofminiature end-plate potentials even in Ca2+-free solutions (Miledi & Thies, 1971); similarly, ouabain (Vizi, 1972(Vizi, , 1977Baker & Crawford, 1975;Meyer & Cooper, 1981; Vyas & Marchbanks, 1981) and veratridine (Sandoval, 1980;Meyer & Cooper, 1981; Vyas & Marchbanks, 1981;Schoffelmeer & Mulder, 1983) can enhance transmitter release in the absence of external Ca +, thus querying the exclusive role of external Ca2+ entry in the depolarization-induced transmitter release.…”

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

“…Several interpretations have been proposed, among them the view that release is a consequence of an increase in the permeability of the membrane, directly associated with the inhibition of Na+-K+-ATPase (Vizi, 1978), or that the increased release is a consequence of depolarization and of a detergent action of ouabain (Vyas & Marchbanks, 1981), or that the opening of (hypothetical) ACh gates is caused by the change in intracellular H+ concentration after the Na+-K+-ATPase had been inhibited (Meyer & Cooper, 1981). Our experiments in which ouabain increased the release of ACh from the innervated diaphragms by 77 % while not affecting that from denervated diaphragms may have important implications regarding the mechanism of the ACh-releasing effect of ouabain.…”