DYNAMIC STORAGE OF DOPAMINE IN RAT-BRAIN SYNAPTIC VESICLES IN-VITRO

The dynamics of catecholamine storage were studied in highly purified, small synaptic vesicles from rat brain both during active uptake or a fter inhibiting uptake with reserpine, tetrabenazine, or removal of ex ternal dopamine. To assess turnover during active uptake, synaptic ves icles were allowed to accumulate [H-3]dopamine ([H-3]DA) for similar t o 10 min and then diluted 20-fold into a solution containing unlabeled DA under conditions such that active uptake could continue. After dil ution, [H-3]DA was lost with single exponential kinetics at a half-tim e of similar to 4 min at 30 degrees C in 8 mM Cl- medium, in which bot h voltage and H+ gradients are present in the vesicles. In 90 mM Cl- m edium, in which high H+ and Cl- gradients but no voltage gradient are present, [H-3]DA escaped at a half-time of similar to 7 min. In both h igh and low Cl- media, similar to 40% of [H-3]DA efflux was blocked by reserpine or tetrabenazine. The residual efflux also followed first-o rder kinetics. These results indicate that two efflux pathways were pr esent, one dependent on DA uptake (and thus on the presence of externa l DA) and the other independent of uptake, and that both pathways func tion regardless of the type of electrochemical HC gradient in the vesi cles. The presence of both uptake-dependent and -independent efflux wa s observed in experiments using DA-free medium, instead of uptake inhi bitors, to prevent uptake. Uptake-independent efflux showed molecular selectivity for catecholamines; [C-14]DA was lost about three times fa ster than [H-3] norepinephrine after adding tetrabenazine directly (wi thout dilution) to vesicles that had taken up comparable amounts of ea ch amine. In addition, the first-order rate constant for uptake-indepe ndent efflux showed little change over a 60-fold range of internal DA concentrations, which suggests that this pathway had a high transport capacity. All efflux was blocked at 0 degrees C, suggesting that efflu x did not occur through a large pore. There was little or no change in the proton gradient in synaptic vesicles, monitored by [C-14]methylam ine equilibration, during the experimental manipulations used here. Th us, the driving force for catecholamine uptake remained approximately constant. The physiological role of uptake-independent efflux could be to allow the monoamine content of synaptic vesicles to be regulated o ver a time range of minutes and, thereby, control the amount released by exocytosis. These results imply that catecholamines turn over with a half-time of minutes during active uptake by brain synaptic vesicles in vitro.