By using electrophysiological and microfluorimetric methods, we found that leech Retzius neurons swell after inhibition of the Na(+)-K(+) pump by the cardiac glycoside ouabain. To explore the mechanism of this swelling, we measured the effect of ouabain on [Na(+)](i), [K(+)](i), and [Cl(-)](i), as well as on the membrane potential, by applying triple-barrelled ion-sensitive microelectrodes. As shown previously, ouabain induced a marked [Na(+)](i) increase, a [K(+)](i) decrease, and a membrane depolarization, and it also evoked an increase in [Cl(-)](i). The analysis of the data revealed a net uptake of NaCl, which quantitatively explained the ouabain-induced cell swelling. In the absence of extracellular Na(+) or Cl(-), NaCl uptake was excluded, and the cell volume remained unaffected. Likewise, NaCl uptake and, hence, cell swelling did not occur when the Na(+)-K(+) pump was inhibited by omitting bath K(+). Also, in K(+)-free solution, [Na(+)](i) increased and [K(+)](i) dropped, but [Cl(-)](i) slightly decreased, and after an initial, small membrane depolarization, the cells hyperpolarized for a prolonged period. It is concluded that the ouabain-induced NaCl uptake is caused by the depolarization of the plasma membrane, which augments the inwardly directed electrochemical Cl(-) gradient.

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In leech P neurons the inhibition of the Na(+)-K(+) pump by ouabain or omission of bath K(+) leaves the membrane potential unaffected for a prolonged period or even induces a marked membrane hyperpolarization, although the concentration gradients for K(+) and Na(+) are attenuated substantially. As shown previously, this stabilization of the membrane potential is caused by an increase in the K(+) conductance of the plasma membrane, which compensates for the reduction of the K(+) gradient. The data presented here strongly suggest that the increased K(+) conductance is due to Na(+)-activated K(+) (K(Na)) channels. Specifically, an increase in the cytosolic Na(+) concentration ([Na(+)](i)) was paralleled by a membrane hyperpolarization, a decrease in the input resistance (R(in)) of the cells, and by the occurrence of an outwardly directed membrane current. The relationship between R(in) and [Na(+)](i) followed a simple model in which the R(in) decrease was attributed to K(+) channels that are activated by the binding of three Na(+) ions, with half-maximal activation at [Na(+)](i) between 45 and 70 mM. At maximum channel activation, R(in) was reduced by more than 90%, suggesting a significant contribution of the K(Na) channels to the physiological functioning of the cells, although evidence for such a contribution is still lacking. Injection experiments showed that the K(Na) channels in leech P neurons are also activated by Li(+).