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Buchtal and collaborators reported thirty years ago an excitatory action of low (10 −3 M) concentrations of ATP on frog muscle, as well as in increase in the sensitivity of the muscle to ACh. These effects have been re-investigated employing both intra-and extra-cellular recording and the technique of iontophoretic drug application. ATP at a concentration of 10 −4 M decreases the recorded resting potential by about 35%. The depolarizing action of ATP is more pronounced in the tibial end of the frog sartorius muscle than in the nerve free pelvic end. In addition ATP, added to the bath and electro-osmotically applied, increases the depolarizing action of ACh. This potentiating effect is particularly marked in denervated muscles.

Succinic anhydride (SA) dissolved in Ringer solution buffered with N-tris (hydroxymethyl) methyl-2-aminoethane sulphonic acid (SA-TES solution) potentiates the depolarizing action of acetylcholine (ACh, 10-40 microM) on frog muscle and the tension induced by bath application of this agonist. Applied from one side of a double-barrelled micropipette, SA-TES increases the amplitude of iontophoretically elicited ACh potentials. The potentiation of the effects of ACh by SA-TES does not involve changes in either the activity of the ACh esterase or the input resistance of the muscle membrane. For depolarizations of frog sartorius muscle, dose-response relationships obtained for ACh concentrations from 0.5 to 20 microM indicate that SA-TES increases the apparent affinity of ACh by a factor of 3. SA-TES exerts an "accelerating' effect on the responses elicited by bath-applied ACh; i.e., it increases the rate of depolarization when ACh is added to the bath and the rate of repolarization upon washing out. These effects are particularly marked in preparations treated with neostigmine (3 microM). SA-TES does not potentiate the depolarizing action of agonists which do not contain an ester group. Moreover, the time course of the responses elicited by these compounds is not influenced by SA-TES. SA-TES fails to influence significantly the effects of the neurally released transmitter. Only a 10% increase in the average amplitude of the endplate potentials was observed. SA hydrolyzes in about 30 min at room temperature; however the SA-TES solution retains its activity for several weeks. Succinate is inactive, and so is SA in Ringer buffered with phosphate. The SA-TES solution contains seven succinyl-TES derivatives, which were separated by ion-exchange chromatography and paper chromatography. At concentrations between 1 to 150 microM, these succinyl-TES derivatives affected the ACh-induced contraction of frog rectus abdominus muscle. The most abundant derivative potentiated the action of high doses of ACh, but was inhibitory at lower ones. The other derivatives were mostly inhibitory. These results are discussed in terms of two hypotheses. One postulates the presence of a diffusion barrier formed by groups that bind ACh and are saturated by SA-TES. The other assumes that SA-TES acts directly on the ACh receptor exerting its potentiating effect through a cooperative mechanism.

INACTIVATION of acetylcholinesterase (AChE) causes the half-decay times of the endplate currents (e.p.cs) and miniature e.p.cs (m.e.p.cs) to increase from about 1.5–2.0ms to 3.0–10.00 ms, times far too long to be accounted for by removal of the transmitter by free diffusion1–3. To explain this, Katz and Miledi1 postulated that, when the AChE is blocked, each acetylcholine (ACh) molecule undergoes numerous collisions with the post-synaptic membrane, leading to several attachments with the ACh receptors and a consequent delay in transmitter removal. This was supported by the observation that ( + )-tubocurarine, which reduces the number of free receptors, markedly decreases the half-decay of m.e.p.cs and e.p.cs after treatment with neostigmine1,3. In addition to this delaying mechanism, due to interaction with the ACh receptors the results described below suggest the existence of a second diffusion barrier for the transmitter at the frog neuromuscular junction. This barrier seems to be formed by non-depolarising sites which bind the ester moiety of ACh. Saturation of these sites with substances which possess –CO–O– or –CO–O–CO– groups accelerates the diffusion of externally applied ACh towards the ACh receptor, enhancing its depolarising effects. The barrier also hinders the outward diffusion of neurally released ACh in neostigmine-treated muscles, but this effect can only be demonstrated if the motor nerve is stimulated at high frequencies (200 Hz).