Uptake of tyramine into synaptic vesicles of the caudate nucleus

Summary

Synaptic vesicles isolated from the caudate nucleus of the pig were preincubated with reserpine, serotonin or tyramine at concentrations which caused a 90% inhibition of the ATP-Mg²⁺-dependent uptake of ¹⁴C-dopamine.

The preincubated vesicles were sedimented by centrifugation and resuspended in drug-free buffer. The vesicles were incubated with ¹⁴C-dopamine in the presence or in the absence of ATP-Mg²⁺; at the end of the incubation period they were separated from the incubation medium by centrifugation. The inhibitory effect of reserpine on the ATP-Mg²⁺-dependent uptake of ¹⁴C-dopamine was slightly diminished, that of serotonin reduced while the inhibitory effect of tyramine was abolished. Hence, tyramine seems to be more easily removable than the other substances.

In another series of experiments the vesicles were incubated with ³H-tyramine and immediately separated by filtration through membrane filters. Addition of ATP-Mg²⁺ enhanced the uptake of ³H-tyramine. The Km of the ATP-Mg²⁺-dependent uptake of ³H-tyramine was 1.4×10⁻⁷ M, the Vmax 29.2 pmoles/mg protein/min. At 0°C the uptake of ³H-tyramine in the absence of ATP-Mg²⁺ was reduced, that in the presence of ATP-Mg²⁺ abolished. Incubation of the vesicles with ³H-dopamine revealed K _m and V _max values similar to those previously found when the vesicles were isolated from the incubation medium by centrifugation. Incubation in the presence of reserpine inhibited the ATP-Mg²⁺-dependent uptake of ³H-tyramine (IC₅₀ 1.4×10⁻⁸ M) and ³H-dopamine (IC₅₀ 4.1×10⁻⁸ M). The results demonstrate that tyramine is taken up into the vesicles by a process which is dependent on ATP-Mg²⁺ and temperature.

Failure of previous attempts to prove an ATP-Mg²⁺-dependent uptake of tyramine when the vesicles were separated by centrifugation seems to be due to release of the accumulated tyramine during the centrifugation procedure.