Metabolism and efflux of [3H]dopamine in rat neostriatum: presynaptic origin of 3,4-[3H]dihydroxyphenylacetic acid
Cubeddu, L.X.; Hoffmann, I.S.; Ferrari, G.B.
Journal of Pharmacology and Experimental Therapeutics 209(2): 165-175
1979
ISSN/ISBN: 0022-3565 PMID: 255152 Document Number: 154206
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 fractions obtained were: 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 neostriatal slices were incubated with [3H]DA (0.83 .mu.M) for 30 min followed by a washout period with amine-free solution. Once the efflux exhibited a single exponential decline (80-170 min of washout), the metabolism pattern found in the medium was 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 Km 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-benzo(a)quinolizine] (BQZ) (30 nM), induced a 4-fold increase in the efflux of radioactive products 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 but the proportion of [3H]OMDA was increased (12.4 .+-. 1.6% controls; 27.6 .+-. 3.5% after washout of BQZ). Cocaine (10 .mu.M) 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 3H efflux induced by BQZ was found as unchanged 3H-amine. Apparently, 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. 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 Km 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 Km values for the acid-DA-metabolites are not due to a probenecid-sensitive transport mechanism. The metabolites of [3H]DA in the medium (efflux) reflected those present in the tissue. The proportions of OMDA and DOPAC were similar in tissue and medium during and after exposure to drugs. Apparently measurements of either tissue or medium DA metabolites equally reflect the preferential metabolic pathway of the neurotransmitter. This may be related to the similar Km for efflux of the principal DA metabolites.