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PKC? Inhibitors Attenuate Amphetamine-Stimulated Dopamine Efflux.

ABSTRACT: Amphetamine abuse afflicts over 13 million people, and there is currently no universally accepted treatment for amphetamine addiction. Amphetamine serves as a substrate for the dopamine transporter and reverses the transporter to cause an increase in extracellular dopamine. Activation of the beta subunit of protein kinase C (PKC?) enhances extracellular dopamine in the presence of amphetamine by facilitating the reverse transport of dopamine and internalizing the D2 autoreceptor. We previously demonstrated that PKC? inhibitors block amphetamine-stimulated dopamine efflux in synaptosomes from rat striatum in vitro. In this study, we utilized in vivo microdialysis in live, behaving rats to assess the effect of the PKC? inhibitors, enzastaurin and ruboxistaurin, on amphetamine-stimulated locomotion and increases in monoamines and their metabolites. A 30 min perfusion of the nucleus accumbens core with 1 ?M enzastaurin or 1 ?M ruboxistaurin reduced efflux of dopamine and its metabolite 3-methoxytyramine induced by amphetamine by approximately 50%. The inhibitors also significantly reduced amphetamine-stimulated extracellular levels of norepinephrine. The stimulation of locomotor behavior by amphetamine, measured simultaneously with the analytes, was comparably reduced by the PKC? inhibitors. Using a stable isotope label retrodialysis procedure, we determined that ruboxistaurin had no effect on basal levels of dopamine, norepinephrine, glutamate, or GABA. In addition, normal uptake function through the dopamine transporter was unaltered by the PKC? inhibitors, as measured in rat synaptosomes. Our results support the utility of using PKC? inhibitors to reduce the effects of amphetamine.

SUBMITTER: Zestos AG

PROVIDER: S-EPMC4909528 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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PKCβ Inhibitors Attenuate Amphetamine-Stimulated Dopamine Efflux.

Zestos Alexander G AG Mikelman Sarah R SR Kennedy Robert T RT Gnegy Margaret E ME

ACS chemical neuroscience 20160328 6

Amphetamine abuse afflicts over 13 million people, and there is currently no universally accepted treatment for amphetamine addiction. Amphetamine serves as a substrate for the dopamine transporter and reverses the transporter to cause an increase in extracellular dopamine. Activation of the beta subunit of protein kinase C (PKCβ) enhances extracellular dopamine in the presence of amphetamine by facilitating the reverse transport of dopamine and internalizing the D2 autoreceptor. We previously d ...[more]

PMID: 26996926

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Project description:Amphetamine (AMPH) elicits its behavioral effects by acting on the dopamine (DA) transporter (DAT) to induce DA efflux into the synaptic cleft. We previously demonstrated that a human DAT construct in which the first 22 amino acids were truncated was not phosphorylated by activation of protein kinase C, in contrast to wild-type (WT) DAT, which was phosphorylated. Nonetheless, in all functions tested to date, which include uptake, inhibitor binding, oligomerization, and redistribution away from the cell surface in response to protein kinase C activation, the truncated DAT was indistinguishable from the full-length WT DAT. Here, however, we show that in HEK-293 cells stably expressing an N-terminal-truncated DAT (del-22 DAT), AMPH-induced DA efflux is reduced by approximately 80%, whether measured by superfusion of a population of cells or by amperometry combined with the patch-clamp technique in the whole cell configuration. We further demonstrate in a full-length DAT construct that simultaneous mutation of the five N-terminal serine residues to alanine (S/A) produces the same phenotype as del-22-normal uptake but dramatically impaired efflux. In contrast, simultaneous mutation of these same five serines to aspartate (S/D) to simulate phosphorylation results in normal AMPH-induced DA efflux and uptake. In the S/A background, the single mutation to Asp of residue 7 or residue 12 restored a significant fraction of WT efflux, whereas mutation to Asp of residues 2, 4, or 13 was without significant effect on efflux. We propose that phosphorylation of one or more serines in the N-terminus of human DAT, most likely Ser7 or Ser12, is essential for AMPH-induced DAT-mediated DA efflux. Quite surprisingly, N-terminal phosphorylation shifts DAT from a "reluctant" state to a "willing" state for AMPH-induced DA efflux, without affecting inward transport. These data raise the therapeutic possibility of interfering selectively with AMPH-induced DA efflux without altering physiological DA uptake.

Dataset Information

PKC? Inhibitors Attenuate Amphetamine-Stimulated Dopamine Efflux.

Publications

PKCβ Inhibitors Attenuate Amphetamine-Stimulated Dopamine Efflux.

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