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.