Amphetamine reduces vesicular dopamine content in dexamethasone-differentiated PC12 cells only following l-DOPA exposure

Amphetamine (AMPH) increases brain dopamine (DA) levels via reversal of the membrane DA transporter. Additional mechanisms have been suggested, including inhibition of vesicular monoamine transporters and vesicular leakage of DA and Ca²⁺. According to the widely-accepted weak base theory, AMPH disrupts the proton gradient required for filling vesicles with DA. As a result, DA and Ca²⁺ will leak from vesicles, giving rise to exocytosis of less-filled vesicles. As several contradictions have been described, the aim of the present study was to re-examine this theory using amperometry and Fura-2 imaging to measure AMPH-induced changes in exocytosis and intracellular Ca²⁺ levels, respectively, in PC12 and chromaffin cells. Unexpectedly, 15 min exposure to AMPH (20-200 μM) does not affect the amount of DA released per vesicle, the frequency of exocytosis or intracellular Ca²⁺ levels in PC12 cells or chromaffin cells. Comparable results were found following prolonged exposure to AMPH (45 min) or at 37°C. When cells were pre-treated with the DA precursor l-DOPA, vesicle content increased to ~150%. When these pre-treated cells are exposed to AMPH, vesicle content is strongly reduced. These results indicate that in dexamethasone-differentiated PC12 cells AMPH-induced vesicle leakage occurs only under specific conditions, therefore arguing for re-evaluation of the theory of AMPH-induced vesicular DA leakage.