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After cessation of repeated, intermittent amphetamine, we detected an emergent Ca 2ϩ -dependent component of amphetamine-induced dopamine release and an increase in calmodulin and Ca 2ϩ -and calmodulin-dependent protein kinase activity in rat striatum. This study examined the involvement of calmodulin-dependent protein kinase II (CaM kinase II) and synaptic vesicles in the enhanced Ca 2ϩ -dependent dopamine release in response to amphetamine or K ϩ in rat striatum. Rats were pretreated for 5 d with 2.5 mg/kg amphetamine or saline and withdrawn from drug for 10 d. The selective CaM kinase II inhibitor KN-93 (1 M), but not the inactive analog KN-92, attenuated the Ca 2ϩ -dependent amphetaminemediated dopamine release from amphetamine-pretreated rats but had no effect in saline-pretreated controls.[ 3 H]Dopamine uptake was unaltered by repeated amphetamine or KN-93 and was Ca 2ϩ independent. Striatal dopamine release stimulated by 50 mM KCl was enhanced twofold after repeated amphetamine compared with that in saline controls but was unaffected by KN-93. To examine the requirement for dopaminergic vesicles in the Ca 2ϩ -dependent dopamine release, we administered reserpine to saline-and amphetamine-pretreated rats 1 d before killing. Reserpine pretreatment did not affect amphetamine-mediated dopamine release from either pretreatment group but completely ablated K ϩ -mediated dopamine release. Reserpine did not disrupt the ability of 1 M KN-93 to block the Ca 2ϩ -dependent amphetamine-mediated dopamine release from amphetamine-pretreated rats. The results indicate that the enhanced dopamine release elicited by amphetamine from chronically treated rats is dependent on Ca 2ϩ -and calmodulin-dependent phosphorylation and is independent of vesicular dopamine storage. On the contrary, the enhanced depolarization-mediated vesicular dopamine release is independent of Ca 2ϩ -and calmodulin-dependent phosphorylation.

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Repeated intermittent amphetamine enhances efflux of dopamine through the dopamine transporter in rat basal ganglia and through the norepinephrine transporter in rat pheochromocytoma PC12 cells. Extracellular Ca 2ϩ is required for the detection of this enhancement in the rat. In this study, we examined the role of Ca 2ϩ and Ca 2ϩ channels in the enhanced amphetamine-induced dopamine efflux that develops in PC12 cells following repeated intermittent amphetamine. Repeated pretreatment of PC12 cells with 1 M amphetamine followed by a drug-free period increased amphetamine-induced efflux of dopamine compared with controls. The enhancement in amphetamine-induced dopamine efflux depended upon the presence of extracellular Ca 2ϩ and was inhibited by the blockade of N-type and L-type Ca 2ϩ channels. The enhanced dopamine efflux was not altered by tetanus toxin or reserpine, treatments that abrogate synaptic vesicle-mediated, exocytotic dopamine efflux. Measurement of intracellular Ca 2ϩ concentrations using fura-2/acetoxymethyl ester revealed that amphetamine increased intracellular Ca 2ϩ by a transporter-dependent mechanism. In amphetamine-pretreated cells, amphetamine elicited a greater increase in intracellular Ca 2ϩ ; this increase depended upon the presence of extracellular Ca 2ϩ and N-and L-type Ca 2ϩ channel activity. The enhanced amphetamine-induced dopamine efflux requires Ca 2ϩ /calmodulin kinase activity. In vehicle-treated cells, 1 M amphetamine inhibited the calmodulin kinase activity although it did not in amphetamine-pretreated cells. This study suggests that repeated intermittent amphetamine couples norepinephrine transporter activity and Ca 2ϩ signaling.

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Repeated intermittent treatment with amphetamine (AMPH) induces both neurite outgrowth and enhanced AMPH-stimulated dopamine (DA) release in PC12 cells. We investigated the role of protein kinases in the induction of these AMPHmediated events by using inhibitors of protein kinase C (PKC), mitogen activated protein kinase (MAP kinase) or protein kinase A (PKA). PKC inhibitors chelerythrine (100 nM and 300 nM), Ro31-8220 (300 nM) and the MAP kinase kinase inhibitor, PD98059 (30 lM) inhibited the ability of AMPH to elicit both neurite outgrowth and the enhanced AMPH-stimulated DA release. The direct-acting PKC activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA, 250 nM) mimicked the ability of AMPH to elicit neurite outgrowth and enhanced DA release. On the contrary, a selective PKA inhibitor, 100 lM Rp-8-Br-cAMPS, blocked only the development of AMPH-stimulated DA release but not the neurite outgrowth. Treatment of the cells with acute AMPH elicited an increase in the activity of PKC and MAP kinase but not PKA. These results demonstrated that AMPH-induced increases in MAP kinase and PKC are important for induction of both the enhancement in transporter-mediated DA release and neurite outgrowth but PKA was only required for the enhancement in AMPH-stimulated DA release. Therefore the mechanisms by which AMPH induces neurite outgrowth and the enhancement in AMPH-stimulated DA release can be differentiated.