Protein Kinase C Inhibitors Block Amphetamine-Mediated Dopamine Release in Rat Striatal Slices 1

The stimulant drug amphetamine is postulated to enhance dopamine release through the plasmalemmal dopamine transporter by an exchange diffusion with synaptosomal dopamine. Because protein kinase C has been shown to have an effect on dopamine transporter activity, we examined the effect of protein kinase C inhibitors on endogenous dopamine release stimulated by amphetamine in perfused rat striatal slices. At concentrations of 1 mM, the selective protein kinase C inhibitors chelerythrine, Ro31–8220 and calphostin C nearly completely inhibited endogenous dopamine release elicited by 1 mM amphetamine. The inactive analog bisindoylmaleimide V had no effect. Extracellular Ca was not required for the effect of the inhibitors. The importance of vesicular dopamine release was examined by determining inhibitor activity in reserpine-treated rats. Dopamine release elicited by 1 mM amphetamine was not significantly altered in reserpine-treated rats compared with control animals. Ro31–8220 at 1 mM completely blocked amphetamine-induced dopamine release in reserpine-treated rats. Activation of protein kinase C with 250 nM of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate increased dopamine release, and the release was not additive with 1 mM amphetamine. Both chelerythrine and Ro31–8220 at 1 mM increased [H]dopamine uptake by 17% and 30%, respectively, whereas a brief exposure to 12-O-tetradecanoylphorbol 13-acetate slightly inhibited [H]dopamine uptake. Our results suggest that amphetamine-mediated dopamine release through the plasmalemmal transporter is highly dependent on protein kinase C activity. AMPHs are highly reinforcing, widely abused stimulant drugs. In humans, amphetamine increases motor and speech activity, decreases appetite and fatigue and elicits euphoria. After a single large dose or after repeated use, a psychosis can develop that resembles paranoid schizophrenia (Angrist, 1994). AMPH increases locomotor and stereotyped behaviors, decreases food intake and leads to self-administration in human as well as animal models (Seiden et al., 1993). Release of DA from the striatum and nucleus accumbens is believed to be an important mediator of AMPH-induced stereotyped (Creese and Iversen, 1974) and locomotor behaviors (Kelly et al., 1975; Kelly and Moore, 1976), respectively. AMPH releases DA through a non-exocytotic, Ca-independent mechanism that requires the presence of the plasmalemmal DA transporter (Raiteri et al., 1979; Kamal et al., 1981; Giros et al., 1996). AMPH both blocks uptake and enhances release of DA by binding to the DA transporter and being transported into the nerve terminal. The prevailing hypothesis is that AMPH releases DA by means of an exchange diffusion mechanism (Fischer and Cho, 1979; Raiteri et al., 1979) involving the reverse of the uptake process. The subsequent dissociation of AMPH after transport into the terminal would increase the availability of the transport carrier to bind and transport DA outside of the neuron (Fischer and Cho, 1979). There have been few studies on the effects of signal transduction mechanisms on AMPH-induced DA release. The treatment of striatal synaptosomes or cultured cells containing the DA transporter with phorbol esters or lipids, which activate the Caand lipid-dependent PKC, decrease [H] DA uptake, suggesting that PKC activation could impair the action of AMPH (Copeland et al., 1996; Huff et al., 1997; Kitayama et al., 1994; Zhang et al., 1997). PKC activation, however, positively affects DA release. Activators of PKC, such as the phorbol ester TPA, diacylglycerol and arachidonic acid, enhance both depolarization-mediated (Brouard et al., 1994; Chandler and Leslie, 1989; L’Hirondel et al., 1995; Pozzan et al., 1984; Shu and Selmanoff, 1988; Versteeg and Ulenkate, 1987; Zurgil and Zisapel, 1985) and basal (Davis and Patrick, 1990) DA release. Conversely, drugs that inhibit PKC were reported to block AMPH-induced [H]DA release from rat striatal synaptoneurosomes (Giambalvo, 1992b). Received for publication June 10, 1997. 1 This work was supported by Grant DA-05066 from the National Institutes for Drug Abuse and NIDA Interdisciplinary Training Grant DA-07267 at the University of Michigan Substance Abuse Research Center (L.K.). ABBREVIATIONS: AMPH, amphetamine; DA, dopamine; DMSO, dimethylsulfoxide; GAP-43, growth-associated protein; HPLC, high-performance liquid chromatography; KRB, Krebs-Ringer buffer; PKC, protein kinase C; TPA, 12-O-tetradecanoylphorbol-13-acetate; ANOVA, analysis of variance. 0022-3565/98/2842-0592$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 284, No. 2 Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 284:592–598, 1998 592 at A PE T Jornals on Sptem er 3, 2017 jpet.asjournals. org D ow nladed from The drugs used in this study, however, were not selective inhibitors of PKC, and the degree of inhibition was not re-