Abstract
Evidence suggests that protein kinase C (PKC) and intracellular calcium are important for amphetamine-stimulated outward transport of dopamine in rat striatum. In this study, we examined the effect of select PKC isoforms on amphetamine-stimulated dopamine efflux, focusing on Ca(2+)-dependent forms of PKC. Efflux of endogenous dopamine was measured in superfused rat striatal slices; dopamine was measured by high performance liquid chromatography. The non-selective classical PKC inhibitor Gö6976 inhibited amphetamine-stimulated dopamine efflux, whereas rottlerin, a specific inhibitor of PKC delta, had no effect. A highly specific PKC beta inhibitor, LY379196, blocked dopamine efflux that was stimulated by either amphetamine or the PKC activator, 12-O-tetradecanoylphorbol-13-acetate. None of the PKC inhibitors significantly altered [3H]dopamine uptake. PKC beta(I) and PKC beta(II), but not PKC alpha or PKC gamma, were co-immunoprecipitated from rat striatal membranes with the dopamine transporter (DAT). Conversely, antisera to PKC beta(I) and PKC beta(II) but not PKC alpha or PKCg amma were able to co-immunoprecipitate DAT. Amphetamine-stimulated dopamine efflux was significantly enhanced in hDAT-HEK 293 cells transfected with PKC beta(II) as compared with hDAT-HEK 293 cells alone, or hDAT-HEK 293 cells transfected with PKCa lpha or PKC beta(I). These results suggest that classical PKC beta(II) is physically associated with DAT and is important in maintaining the amphetamine-stimulated outward transport of dopamine in rat striatum.
Highlights
The plasmalemmal dopamine transporter (DAT)1 is a presynaptic protein that belongs to the SLC6 family of Naϩ/ClϪdependent neurotransmitter transporters
Evidence suggests that protein kinase C (PKC) and intracellular calcium are important for amphetaminestimulated outward transport of dopamine in rat striatum
We report that classical PKC isozymes regulate the mechanism of action of amphetamine in eliciting outward transport of dopamine
Summary
The plasmalemmal dopamine transporter (DAT)1 is a presynaptic protein that belongs to the SLC6 family of Naϩ/ClϪdependent neurotransmitter transporters. The non-selective classical PKC inhibitor Go 6976 inhibited amphetamine-stimulated dopamine efflux, whereas rottlerin, a specific inhibitor of PKC␦, had no effect. A highly specific PKC inhibitor, LY379196, blocked dopamine efflux that was stimulated by either amphetamine or the PKC activator, 12-O-tetradecanoylphorbol-13-acetate.
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