The ability of synaptosomes prepared from rat brain tissue to accumulate pglu-his-[2,3-3H]pro-NH2 ([3H]TRH), a possible peptidergic neurotransmitter, was investigated. The uptake of [3H]-dopamine, a proven neurotransmitter, was evaluated in parallel experiments. When a synaptosome preparation derived from homogenates of male rat hypothalamic or cerebrocortical tissue was diluted with an equal volume of Hanks’ balanced salt solution and incubated with [3H]TRH, uptake of a radiolabeled substance(s) by subcellular particles was demonstrated. Similar results were obtained in mixtures containing [3H]dopamine. The particles accumulating radiolabeled compounds were identified as synaptosomes on the basis of size, ultrastructural profiles, and sedimentation characteristics on continuous and discontinuous sucrose density gradients. The uptake of a radiolabeled compound(s) from incubation media containing [3H]TRH was dependent on temperature, duration of the incubation period, presence of glucose and electrolytes, and structural integrity of the synaptosomes. The tritium-labeled compound isolated from synaptosomes following incubation of the synaptosome preparation with [3H]-TRH was found to be [3H]proline rather than [3H]-TRH. An examination of the tritium-labeled compounds in the medium after incubation revealed extensive metabolism of [3H]TRH. When the metabolism of [3H]TRH during incubation was minimized by the use of purified synaptosome preparations, the uptake of radioactive compound(s) by the synaptosomes was essentially abolished. However, uptake of [3H]dopamine by purified synaptosomes was not impaired, indicating that the synaptosomes had not been damaged during purification. The results of these studies show that, unlike some proved neurotransmitters, [3H]TRH is not taken up by axonal terminals in vitro but that the [3H]TRH metabolite, [3H]proline, is taken up by these sub-neuronal organelles.
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