Studies on the uptake of [³H]thyrotropin-releasing hormone and its metabolites by synaptosome preparations of the rat brain

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.