Dopamine transporter antagonists block phorbol ester-induced dopamine release and dopamine transporter phosphorylation in striatal synaptosomes
Introduction
Activators of protein kinase C, such as 12-O-tetradecanoyl phorbol-13-acetate (TPA), diacylglycerol and arachidonic acid, enhance both depolarization-mediated (Robinson, 1991) and basal release of dopamine Davis and Patrick, 1990, L'hirondel et al., 1995. Protein kinase C inhibitors, however, have little or no effect on Ca2+- or depolarization-evoked release in the absence of protein kinase C activation (see discussion in Robinson, 1991) suggesting that protein kinase C does not contribute to depolarization-evoked dopamine release in the absence of an accompanying stimulus. It has been concluded that protein kinase C is not required for Ca2+-secretion coupling but could modulate release by phosphorylating a key substrate that regulates depolarization (Robinson, 1991, Coffey et al., 1993.
Release of dopamine without depolarization in response to the protein kinase C activators arachidonic acid (L'hirondel et al., 1995), diacylglycerol (Davis and Patrick, 1990) and TPA (Pozzan et al., 1984) has been demonstrated in striatal synaptosomes and PC12 cells. The diacylglycerol-induced dopamine release in rat striatal synaptosomes was demonstrated to be independent of extracellular calcium (Davis and Patrick, 1990). Further, we and Giambalvo (1992) reported that protein kinase C inhibitors blocked the Ca2+-independent amphetamine-mediated release of dopamine from rat striatal slices. Both 1 μM amphetamine and 250 nM TPA released comparable amounts of dopamine but release in the presence of both agents was non-additive (Kantor and Gnegy, 1998). Since amphetamine requires the plasmalemmal dopamine transporter for its releasing activity (Giros et al., 1996), this suggests that protein kinase C is required for amphetamine to elicit reverse transport of dopamine through the transporter. It further suggests that direct protein kinase C activation could elicit reverse transport of dopamine through the dopamine transporter.
The objective of this study was to determine whether protein kinase C activation can induce reverse transport of endogenous dopamine through the dopamine transporter. Using TPA as an activator of protein kinase C, we determined that TPA-mediated release from striatal synaptosomes and slices is specifically due to protein kinase C activation and can be blocked by antagonists of the dopamine transporter.
Section snippets
Preparation of striatal slices
Female Holtzman rats (weight 117–125 g) were killed by decapitation and the striatum was dissected on ice using a brain-cutting block as described (Heffner et al., 1980). The striatal tissue of each rat was sectioned into 1-mm3 pieces and placed into ice-cold Krebs Ringer Buffer (KRB) containing 125 mM NaCl, 2.7 mM KCl, 1.0 mM MgCl2, 1.2 mM KH2PO4, 10 mM glucose, 24.9 mM NaHCO3 and 0.25 M ascorbic acid. The buffer was oxygenated with 95% O2/5% CO2 for 1 h.
Preparation of striatal synaptosomes
A striatal P2 fraction was made from
TPA-induced dopamine release is independent of extracellular Ca2+
Release of dopamine in response to 250 nM and 1 μM TPA is shown in Fig. 1A. As shown in Fig. 1A, TPA elicits a dose-dependent increase in dopamine release from rat striatal synaptosomes. The release appeared to be independent of extracellular calcium since calcium was not present in the KRB. To further demonstrate the calcium independence of the TPA action, cadmium, a blocker of plasmalemmal calcium channels, was included in the KRB. The data in Fig. 1B, expressed as percent baseline,
Discussion
The effect of protein kinase C activation on dopamine transporter activity is a subject of considerable investigation. Protein kinase C activators have been shown to inhibit uptake of dopamine (Kitayama et al., 1994, Copeland et al., 1996, Huff et al., 1997, Zhang et al., 1997. Direct phosphorylation of dopamine transporter by protein kinase C activators has been demonstrated both in dopamine transporter-transfected cultured cells and in striatal synaptosomes. This phosphorylation was
Acknowledgements
Support for these studies came from the Biomedical Research Council, University of Michigan. We would like to thank Dr. Allan Levey (Emory University, Atlanta, GA) for his generous gift of antibody to the plasmalemmal dopamine transporter.
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