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

Abstract

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.

Amphetamine Elicits Opposing Actions on Readily Releasable and Reserve Pools for Dopamine

“…Consistent with region-specific actions of AMPH on vesicular dopamine stores, we have recently shown that AMPH may up-regulate vesicular dopamine release in the ventral striatum by mobilizing the reserve pool but by activating dopamine synthesis and inhibiting dopamine degradation in the dorsal striatum [65]. Different distributions of small, clear and large, dense-core vesicles in the two striatal sub-regions [66] may also contribute to the differential response to AMPH. Clearly, more work needs to be done to resolve the differential depleting effects of AMPH on dopamine vesicles in the dorsal and ventral striatum.…”

The real catecholamine content of secretory vesicles in the CNS revealed by electrochemical cytometry

“…An action at synaptic vesicles has been proposed to occur by displacement of dopamine from synaptic vesicles via a “weak base mechanism” that collapses the acidic vesicular pH gradient and/or interactions with the vesicular transporter34. However, the hypothesis that amphetamine affects synaptic vesicle transmitter stores under pharmacologically relevant conditions remains controversial and it has been reported that amphetamine does not, in fact, redistribute dopamine from vesicles35. The mechanism of amphetamine has also been investigated by performing measurements at PC12 cells and at the dopaminergic nerve of Planorbis corneus 32.…”

DOPA Homeostasis by Dopamine: A Control-Theoretic View

“…The synthesized DA is stored in synaptic vesicles for release. Significant leakage of transmitter molecules out from vesicles has been observed [ 16 , 17 , 18 , 19 , 20 ], and we tentatively assume that leakage also occurs in vivo. Upon arrival of an action, potential vesicles are emptied, and DA is released into the synaptic cleft.…”

“…There is evidence that catecholamine transmitters stored in vesicles leak into the cytoplasm [ 16 , 17 , 18 , 19 , 20 ]. An analysis of the kinetic data by Fried [ 16 ] and Schonn et al [ 17 ] ( Appendix B ) show that the vesicular leakage of DA can be described as a first-order process with respect to (Equation (12)).…”

“…ROS can oxidize DOPA [ 30 ] (outlined in red). Cytosolic DA concentration is increased due to the re-entry of DA (flux ) and by vesicular leakage [ 16 , 17 , 18 , 19 , 20 ] (flux ). Inflow of Ca by neuronal stimulation lead to release of vesicular DA into the synaptic cleft.…”