Release of synaptosomal dopamine formed from tyrosine and l-dopa

Abstract

The present report compares the synaptosomal release of [ 3H]dopamine, continuously forming from [ 3H]DOPA, with that of [ 14C]dopamine, forming from [ 14C]tyrosine as a marker of dopaminergic nerve endings. For the purpose of the comparison, synaptosomal (P 2) preparations from rat caudate nuclei were incubated with l-[ 3H]DOPA and [ 14C]tyrosine for 10 min and the particulates were rapidly separated from the medium postincubation. The separated fractions were analyzed for the level of double labelled ( 14C/ 3H) dopamine and the synaptosomal content of the labelled substrates. Of the total labelled dopamine formed, the fraction that was present in the medium, following the synaptosomal release, was determined. Tested were the release enhancing effects of various additions which included several known dopaminergic agents, serotonin and 5-hydroxytryptophan. The data show that the addition of dopamine to the incubation mixture to either 0.5 or 1.0 μM concentration markedly enhanced the release of labelled dopamine. Serotonin when added to 5.0 μM concentration also raised the medium content of labelled dopamine but it was ineffective at 1.0 μM. 5-Hydroxytryptophan, 1.0 or 5.0 μM, had no effect. For the comparison of the release enhancing effects of an addition of [ 14C]dopamine and [ 3H]dopamine, the corresponding release indices (release index = medium/total ratio of labelled dopamine in the presence of an addition divided by the same ratio in control (no addition) sample) were determined. The data indicate that the index of [ 14C]dopamine did not differ significantly from that of [ 3H]dopamine following the addition of either dopamine, serotonin or 5-hydroxytryptophan at any of the concentrations tested. A similar lack of difference between the index of [ 14C]dopamine and that of [ 3H]dopamine was observed following the addition of reserpine (1.8 μM), although a considerable enhancement of labelled dopamine release occurred. The addition of either amphetamine (9.1 μM) or amfonelic acid (9.1 μM) also enhanced labelled dopamine release but in their presence the index of [ 3H]dopamine was significantly higher than that of [ 14C]dopamine. Amfonelic acid preferentially raised the [ 3H]dopamine index at the lowest concentration of 0.91 μM that we have tested and also after only 5 min of incubation; coaddition of reserpine increased the [ 14C]dopamine release, thus abolishing the preferential effect of amfonelic acid on [ 3H]dopamine. When the incubation was performed without an addition (control sample), no difference (NS) was observed between the particulate to medium distribution of [ 14C]dopamine and that of [ 3H]dopamine after either 5, 10 or 15 min. The results suggest that (a) dopamine synaptosomally formed from l-DOPA may exist in a pool distinct from that dopamine arising from tyrosine hydroxylation and (b) the observed dopamine compartmentation may be due to some mechanism distinct from any possible participation of serotoninergic partivles. Also, the present results support the previously suggested existence of synaptosomal DOPA in pools.