Metabolism and efflux of [³H]dopamine in rat neostriatum: Presynaptic origin of 3,4-[³H]dihydroxyphenylacetic acid

A method for the separation of dopamine (DA) and its metabolites was developed. The procedure is based on the separation of catechols on small alumina columns and the subsequent absorption of the amines to small Dowex columns. The following fractions were obtained: O-methylated-deaminated metabolites (OMDA) which include 3-methoxy-4-hydroxyphenylacetic acid (HVA) and 3-methoxy-4-hydroxyphenylethanol (MOPET); 3-methoxytyramine (MTA); 3,4-dihydroxyphenylethanol (DOPET); DA; and 3,4-dihydroxyphenylacetic acid (DOPAC). The recoveries for all fractions were greater than 85%. About 2% of the DA was found in the DOPAC fraction and less than 1% of the catechol acid was present in the DOPET fraction. With this procedure, the metabolism of [3H]DA in the rat neostriatum was determined. RAT Rat neostriatal slices were incubated with [3H]DA (0.83 μM) for 30 min. This was followed by a washout period with amine-free solution. Once the efflux exhibited a single exponential decline (80-170 min of washout), the following pattern of metabolism was found in the medium: DOPAC: 61.3 ± 0.3%; OMDA: 21.0 ± 0.6%; DA: 10.1 ± 0.3%; MTA: 2.6 ± 0.1%; and DOPET: 1.4 ± 0.1%. The rate constants and the half-times for the efflux of [3H]DOPAC and [3H]OMDA were 0.0485 min-1, 14.3 min and 0.0737 min-1, 9.4 min, respectively. These values were not affected by probenecid (10-5-10-3 M). Ro 4-1284, a fast-acting, reserpine-like agent [2-hydroxy-2-ethyl-3-isobutyl-9,10-dimethoxy-1,2,3,4,6,7-hexahydro-11b-H-henzo(a)quinolizine] (BQZ) (30 nM), induced a 4-fold increase in the efflux of radioactive products. This was due to a marked (9-fold) and selective increase in the efflux of [3H]DOPAC. After removal of the drug, the efflux of total 3H and [3H]DOPAC returned toward control levels; however, the proportion of [3H]OMDA was increased (12.4 ± 1.6% controls; 27.6 ± 3.5% after washout of BQZ). Cocaine per se (10 μM) produced a small increase in the basal efflux of total 3H and [3H]DA and a slight reduction (15%) in that of [3H]DOPAC. In cocaine-treated slices, a greater percentage of the total increase in tritium efflux induced by BQZ was found as unchanged 3H-amine. The results suggest that during resting efflux, and when this is accelerated by exposure to reserpine-like agents, most of the [3H]DOPAC is formed intraneuronally from [3H]DA, probably during its passage from the vesicles to the axonal membrane. In addition, some of the amine which escapes the nerves unmetabolized can be converted to DOPAC at presynaptic sites after being reaccumulated by the neuron through the cocaine-sensitive amine uptake process. The high rate constant for efflux of [3H]DOPAC probably accounts for the fact that rapid changes in its rate of synthesis (exposure to BQZ) are reflected by quick changes in the rate of efflux of this metabolite. The high k values for the acid-DA-metabolites are not due to a probenecid-sensitive transport mechanism. The metabolites of [3H]DA in the medium (efflux) always reflected those present in the tissue. The proportions of OMDA and DOPAC were similar in tissue and medium during and after exposure to drugs. There results indicate that measurements of either tissue or medium DA metabolites equally reflect the preferential metabolic pathway of this neurotransmitter. This could be related to the rather similar rate constants for efflux of the principal DA metabolites.