3H-5-hydroxytryptamine accumulation by rat brain synaptic vesicles in a membrane-impermeant medium, and selective...

In order to examine possible selectivity of amine uptake by synaptic vesicles, the ATP-stimulated accumulation of 3H-5-hydroxytryptamine (5HT) by synaptic vesicles from rat whole brain was examined in a medium comprised largely of membrane-impermeant anions (d-tartrate). Such media have previously been shown to stabilize vesicular accumulation of several neurotransmitters. Accumulation of 3H-5HT did not occur in tartrate medium alone, but was increased biphasically with increasing concentrations of both potassium phosphate and potassium bicarbonate. At optimal concentrations of each anion (10 mM), stable accumulation of 3H-5HT was observed at 37 degrees (26.1 +/- 1.2 pmol/mg protein; Km 6 X 10(-7) M), which was reduced by greater than 95% in the absence of K2ATP, at 4 degrees C, in the presence of 10(-6) M reserpine, or in the presence of the proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Uptake was significantly antagonized by millimolar concentrations of Na+, Mg++ or Cl-, but was unaffected by ouabain (10(-5) M). Pretreatment of animals with 5,7-dihydroxytryptamine (5,7-DHT) (200 micrograms, intraventricular) 10 days prior to sacrifice reduced endogenous 5HT levels by 70%, while levels of endogenous norepinephrine (NE) and dopamine (DA) were unaffected. Accumulation of 3H-5HT, examined in the presence of 10(-6) M NE to block 3H-5HT accumulation by vesicles from noradrenergic nerve endings, was reduced by 40% in vesicles from treated animals. Vesicular accumulation of 3H-(-)-NE and 3H-DA was unaffected by 5,7-DHT treatment. The data suggest the possibility of preferential accumulation of 3H-5HT by vesicles arising from serotonergic nerve endings.