Vesicular monoamine uptake by digitonin-permeabilized PC12 cells: inhibitory effect of neuromodulators and drugs on the amine transport.

Abstract

Vesicular amine transport is crucial for activity of monoaminergic neurons since translocation of cytosolic amine to storage vesicles is required for both exocytotic release and reuptake of the neurotransmitter. We developed a convenient assay system for vesicular amine transport based on permeabilizing the plasma membrane of pheochromocytoma PC12 cells with digitonin at concentrations of 100 to 150 microM. Serotonin (5HT) is a better substrate than either epinephrine or norepinephrine in this assay system. In the presence of 2 mM ATP, 5HT uptake by the permeabilized cells increased linearly for at least 30 min at 25 degrees C, whereas without addition of exogenous ATP, 5HT uptake reached an apparent plateau after 20 min incubation. Reserpine (500 nM) completely blocked the ATP-dependent 5HT uptake in this system whereas nomifensine (10 microM) had no effect, indicating specificity for vesicular transport. Treatment of intact PC12 cells with AMP dose-dependently decreased 5HT uptake with an EC50 of 20-30 microM as measured after cell permeabilization. Treatment of the intact PC12 cells with forskolin and phorbol 12-myristate 13-acetate prior to permeabilization also down-regulated vesicular 5HT transport, whereas the addition of either of these agents into the reaction mixture for the amine uptake by already permeabilized cells did not alter the vesicular uptake activity. Thus, the system can be applied to studies on regulation of the vesicular amine transport by physiological signaling molecules, intracellular messengers, and drugs.