The accumulation of dopamine into striatal synaptosomes, and its inhibition: comparison of crude, partially purified and purified synaptosomal preparations
Tuomisto, J.; Tuomisto, L.
Medical Biology 57(4): 238-245
1979
ISSN/ISBN: 0302-2137 PMID: 513880 Document Number: 147453
Various methods using rat synaptosomes purified to differing degrees were compared to determine their validity for studying the dopamine uptake by striatal synaptosomes. After homogenization in a glass-teflon homogenizer, the Whittaker fractions S1, P2 and B (750 .times. g supernatant, resuspended 17,000 .times. g sediment or synaptosome bands between 0.8 and 1.2 M and/or between 1.2 and 1.3 M sucrose layers after density gradient centrifugation, respectively) were used and incubated for 1-10 min in a Krebs-Henseleit bicarbonate buffer, with various concentrations of [3H] dopamine and with or without various inhibitors. With increasing purification the specific activity of the initial uptake, as measured for the amount of protein, increased 5- to 6-fold. The activity in absolute figures (as given for the amount of striatal tissue used for the preparation) decreased to about 1/6. This was due to the loss of synaptosomes to other fractions during the purification. During the stage of stage of initial uptake, various inhibitors of dopamine accumulation caused a similar decrease in crude and purified preparations. No differences were seen in the uptake kinetics either, with the exception of the predictable decrease in Vmax due to less synaptosomes per sample. During prolonged incubations not representing the stage of initial uptake the accumulation continued for a longer time in more purified preparations. In these conditions reserpine (assumed to inhibit the storage of dopamine rather than block the membrane uptake) caused a substantially greater reduction in the accumulation of dopamine to a certain plateau level in cruder fractions. A plausible contributing factor is substrate metabolism by monoamine oxidase, which is present in free mitochondria in crude fractions. In simple inhibitor studies where the accumulation of dopamine is studied at the stage of initial uptake (i.e., the number of synaptosomes per sample is such that the substrate concentration will not essentially decrease during the incubation; the incubation time is short, at the most 2 min and preferably shorter; and the substrate concentration does not exceed the Km values) selection of the preparation (S1, P2 or B-fraction) does not seem to be critical. In these conditions the effect of the possible releasing properties of the uptake inhibitor can also be minimized.