Chemical modification by maleimide of toxic and nontoxic ouabain actions on neuromuscular transmission in the frog.

A dual action of ouabain on the Mg(2+)-blocked frog neuromuscular transmission was studied in two different experimental procedures by measuring the mean quantal content (m) of endplate potentials evoked during repetitive nerve stimulation. The nontoxic action of low-dose (1 microM) ouabain was observed as a counterclockwise pivoting of the linear stimulation frequency versus the log m relation, termed frequency augmentation-potentiation (FAP), whereas the toxic effect of higher doses (> or = 10 microM) of ouabain was recorded as a slow exponential increase in m. Since neural Na+,K(+)-ATPase consists of two or three isoforms, the nontoxic action of low-dose ouabain was anticipated to be a consequence of the selective inhibition of ouabain-sensitive alpha(+) (alpha 2 and alpha 3 or neural type) Na+,K(+)-ATPase activity. Such a possibility was assessed by applying epsilon-maleimidocaproic acid (MCA), a membrane-impermeant sulfhydryl reagent. MCA 15 microM was found in the present study to suppress the nontoxic effect of ouabain without altering the toxic effect. The results strongly suggest that the toxic and nontoxic actions of ouabain are the result of actions on different entities. The ouabain-sensitive neural type Na+,K(+)-ATPase, which is abundant in neural tissues, seems to play an important role in modulation of transmitter release observable as FAP.