The effect of ouabain on amino acid and orthophosphate influxes in squid giant axons.

1. Some effects of ouabain on the uptake of 14C-labelled amino acids and 32P-labelled orthophosphate into squid axons have been investigated. 2. Ouabain in artificial sea water reduced the uptake of glycine, L-alanine, L-arginine, L-aspartate and L-glutamate. After a period of 3 hr in ouabain the inhibition of glycine and L-glutamate uptake was appreciable. 3. After 3 hr in ouabain the axoplasmic Na concentration had increased by only about 14% and the K concentration had fallen by only about 9%. It therefore seems unlikely that changes in the Na and K gradients across the membrane due to ouabain are the reason for the inhibition of amino acid uptake. This was confirmed when the axons were made to conduct impulses at 200/sec for 40 min. There was no subsequent inhibition of glycine and L-glutamate uptake, even though the axoplasmic Na increased by about 42% and the K decreased by about 37%. 4. A detailed investigation of the time course of the inhibition of amino acid uptake by ouabain in which intracellular scintillator techniques were used, showed that there was no inhibition for 30--100 min, and then it occurred fairly quickly. This course of events was not affected by the protein synthesis inhibitor cycloheximide. It is unlikely that the inhibition of orthophosphate uptake is subject to a similar delay since this had reached a maximum after 40 min. 5. It is concluded that the inhibitory effects of ouabain on amino acid uptake in squid axons are not due to effects on the Na and K gradients brought about by inhibition of the Na-K pump. The delay in the inhibiton may reflect the operation of an intracellular regulatory mechanis, which is initiated when ouabain inhibits the pump but which takes time to operate. If the formation of an inhibitory second messenger molecule is involved this is unlikely to be a protein.