(+)-Tubocurarine, hexamethonium, atropine, ouabain, and removal of potassium from the bathing medium were examined for their effects on indirectly evoked twitches (IT) of mouse phrenic nerve-diaphragm muscles in the presence or absence of neostigmine. 2. Neostigmine increased the amplitude of IT. The twitch potentiation was reduced by (+)-tubocurarine at low concentrations that had no inhibitory effect on normal IT. Hexamethonium (10-100 microM), but not atropine (0.1-1 microM), partially inhibited the twitch potentiation. Neither hexamethonium nor atropine had an inhibitory effect on IT in the absence of neostigmine. 3. Ouabain (5 microM) abolished the twitch potentiation by neostigmine while having no inhibitory effect on directly evoked twitches in the presence of neostigmine and (+)-tubocurarine together. 4. The potentiating effect of neostigmine was less in a potassium-free bathing solution. The inhibitory effect of ouabain disappeared in this solution. 5. Reinclusion of KCl at 2.5 mM restored both the potentiating effect of neostigmine and the antagonistic effect of ouabain. This reinclusion did not potentiate IT in the absence of neostigmine. 6. An interaction resembling that between ouabain and neostigmine was obtained between ouabain and physostigmine or paraoxon. 7. Both endplate potentials (e.p.ps) and miniature e.p.ps increased in terms of their amplitude and duration in the presence of neostigmine. Ouabain did not reduce the enhanced endplate responses. 8. These results indicate that the potentiation of IT by anticholinesterases may occur via nicotinic receptors which are sensitive to both (+)-tubocurarine and hexamethonium, and that the interaction between anticholinesterases and ouabain depends on the presence of K+. It appears that the mechanisms of twitch potentiation are dependent on the ionic gradients maintained by Na+-K+-ATPase.
