Enhanced amphetamine- and K+-mediated dopamine release in rat striatum after repeated amphetamine: differential requirements for Ca2+- and calmodulin-dependent phosphorylation and synaptic vesicles

Abstract

After cessation of repeated, intermittent amphetamine, we detected an emergent Ca2+-dependent component of amphetamine-induced dopamine release and an increase in calmodulin and Ca2+- 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 Ca2+-dependent dopamine release in response to amphetamine or K+ 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 Rats were pretreated for 5 d with 2.5 mg/kg amphetamine or saline and withdrawn from drug for 10 d. The selective CaM kinase II inhibitor KN-93 (1 microM), but not the inactive analog KN-92, attenuated the Ca2+-dependent amphetamine-mediated dopamine release from amphetamine-pretreated rats but had no effect in saline-pretreated controls. [3H]Dopamine uptake was unaltered by repeated amphetamine or KN-93 and was Ca2+ 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 Ca2+-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+-mediated dopamine release. Reserpine did not disrupt the ability of 1 microM KN-93 to block the Ca2+-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 Ca2+- and calmodulin-dependent phosphorylation and is independent of vesicular dopamine storage. On the contrary, the enhanced depolarization-mediated vesicular dopamine release is independent of Ca2+- and calmodulin-dependent phosphorylation.

Figures

Fig. 1.

AMPH-mediated DA release in AMPH- and saline-pretreated rats. Female Holtzman rats were pretreated with saline (triangles) or AMPH (squares) as described in Materials and Methods. DA release from the striatal slices was measured in response to AMPH (closed symbols) or KRB (open symbols) as described in Materials and Methods and reported as picomoles of DA per milligram wet weight (ww) ± SEM. Drugs given as a 2.5 min bolus at fraction 7 will reach the slices at fraction 9 (n = 3).A–A and S–A represent DA release from AMPH (A)- and saline (S)-pretreated rats after perfusion with 1 μm AMPH. A–KRB and S–KRBindicate basal DA levels from striatal slices from A- and S-pretreated rats. By the use of ANOVA to compare values for fraction 10, the peak DA responses differ (p < 0.0001). In post hocTukey–Kramer tests, S–KRB and A–KRBdiffer from S–A at p < 0.05 andA–A at p < 0.001.S–A differs from A–A atp < 0.001.

Fig. 2.

The effect of KN-93 and KN-92 on AMPH-mediated DA release in AMPH- and saline-pretreated rats. Rats were given repeated saline (A, C) or AMPH (B,D), and striatal slices were analyzed for DA release as described in Materials and Methods. DA release in striatal slices was measured in response to AMPH (closed symbols) or KRB (open symbols) in the absence (squares) or presence (diamonds) of 10 μm KN-93 or 10 μm KN-92. Results are reported as picomoles of DA per milligram wet weight (ww) ± SEM (n= 3). A, C, The effect of KN-93 or KN-92 on basal and AMPH-mediated DA release in saline-pretreated rats.B, D, The effect of KN-93 or KN-92 on AMPH-mediated DA release from AMPH-pretreated rats. Statistical analyses were performed using a one-way ANOVA with Tukey–Kramerpost hoc analysis on the peak AMPH response, fraction 10 (n = 3). A, C, ANOVA,p = 0.01; p < 0.05 for AMPH (A) compared with KN-93 (KN93) or KN-92 (KN92) alone and KRB.B, ANOVA, p < 0.0001;p < 0.001 for KRB andKN93 alone compared with A;p < 0.05 for KRB andKN93 alone compared with KN93+A; andp < 0.01 for A compared withKN93+A. D, ANOVA, p< 0.001; p < 0.01 for KRB andKN92 alone compared with A andKN92+A.

Fig. 3.

K+-mediated DA release from AMPH- and saline-pretreated rats. Rats were given repeated saline (triangles) or AMPH (squares) as described in Materials and Methods. DA release from the striatal slices was measured in response to 50 mm KCl (closed symbols) or KRB (open symbols) as described in Materials and Methods and was reported as picomoles of DA per milligram wet weight (ww) ± SEM.A–K+ andS–K+ represent AMPH (A)- and saline (S)-pretreated rats given a bolus of 50 mm KCl, whereas A–KRB andS–KRB indicate basal DA levels from striatal slices from A- and S-pretreated rats. In comparing the peak K+ response,p < 0.03 forA–K+ compared withS–K+ (Student’s ttest; n = 6).

Fig. 4.

The effect of KN-93 on K+-mediated DA release from AMPH- and saline-pretreated rats. Rats were given repeated saline (A) or AMPH (B) as described in Materials and Methods. DA release from the striatal slices was measured in response to 50 mm KCl (closed symbols) or KRB (open symbols) in the absence (squares) or presence (diamonds) of 10 μm KN-93. Results are reported as picomoles of DA per milligram wet weight (ww) ± SEM (n= 3). Statistical analyses were performed using a one-way ANOVA with Tukey–Kramer post hoc analysis on the peak AMPH response, fraction 10. A, ANOVA, p< 0.03; all values for K+ differed from those for KRB at p < 0.05. B, ANOVA,p < 0.002; values for K+differed from those for KRB at p < 0.01. In neither A nor B did values with KN-93 (KN93) differ from their respective values without KN-93.

Fig. 5.

The effect of Ca2+ and KN-93 on [H3]DA uptake in AMPH- and saline-pretreated rats. Striatal slices from saline (light gray)- and AMPH (dark gray)-pretreated rats were incubated as indicated. Values in the left-hand set ofbars are those obtained when striata were incubated in KRB lacking CaCl2. All other samples were incubated in Ca2+-containing KRB. [3H]DA uptake was measured as described in Materials and Methods. In some experiments, slices were pretreated for 15 min with 10 μmKN-93, 10 μm nomifensine (NF), or 5 μm GBR-12935 (GBR). The results are expressed as femtomoles of DA per milligram wet weight (ww) of slices. Results represent the average ± SEM of three separate experiments. All values with nomifensine and GBR-12935 differed from values without these drugs atp < 0.05.

Fig. 6.

The effect of reserpine on AMPH- and K+-mediated DA release in saline- and AMPH-pretreated rats. Rats received repeated saline or AMPH as described in Materials and Methods. On the ninth day after cessation of repeated saline or AMPH, rats were treated with either vehicle or reserpine (5 mg/kg, s.c.) such that four groups were formed: saline–vehicle (S), saline–reserpine (rS), AMPH–vehicle (A), and AMPH–reserpine (rA). Striatal slices were prepared from rats of each group, and DA release was measured in response to AMPH (A), 50 mm KCl (K+; B), or continued KRB (basal release; open square). KRB values in Aand B comprise the average of basal DA release data from saline, AMPH, and reserpine pretreatment that were all the same. Results are reported as picomoles of DA per milligram wet weight (ww) ± SEM (n = 6). Statistical analyses were performed using a one-way ANOVA with Tukey–Kramerpost hoc analysis on the peak AMPH response, fraction 10 (n = 3). A, DA release in response to AMPH (filled symbols) in saline- or AMPH-pretreated rats given vehicle (solid lines) or reserpine (dashed lines) the day before being killed (ANOVA, p < 0.0001). In post hocTukey–Kramer analysis, all values differed from KRB atp < 0.01, and all saline pretreatments (S–A and rS–A) differed from all AMPH pretreatments (A–A and rA–A) atp < 0.01. No group with reserpine pretreatment differed from the corresponding group without reserpine pretreatment (S–A vs rS–A) and (A–Avs rA–A). B, DA release in response to K+ (filled symbols) in saline- or AMPH-pretreated rats given vehicle (solid lines) or reserpine (dashed lines) the day before being killed (ANOVA, p < 0.0001). In post hocTukey–Kramer analysis, values from vehicle-treated rats (S–K+ andA–K+) differed from KRB values atp < 0.001 and from K+ values for reserpine-treated rats at p < 0.01. Values from reserpine-pretreated rats did not differ from KRB values (baseline).

Fig. 7.

The effect of KN-93 on AMPH-mediated DA release in rats treated with reserpine after previous saline and AMPH pretreatment. Rats received repeated injections of saline (A) or AMPH (B) and were treated with reserpine on withdrawal day 9 as described in Materials and Methods and the legend to Figure 6. DA release in striatal slices was measured in response to AMPH (closed symbols) or KRB (open symbols) in the absence (squares) or presence (diamonds) of 10 μm KN-93. Results are reported as picomoles of DA per milligram wet weight (ww) ± SEM (n = 3). Statistical analyses were performed using a one-way ANOVA with Tukey–Kramerpost hoc analysis on the peak AMPH response, fraction 10. A, The effect of KN-93 on basal and AMPH (A)-mediated DA release in saline (S)- and reserpine-pretreated rats. ANOVA,p < 0.0001; values with AMPH differed from all values without AMPH at p < 0.001; values with KN-93 (KN93) were no different from that of the comparable control. B, The effect of KN-93 on AMPH-mediated DA release from AMPH- and reserpine-pretreated rats. ANOVA, p < 0.0001; all values with amphetamine were different from those without AMPH at p < 0.01; value for AMPH + KN-93 differed from that for AMPH alone (KN93+A vs A) at p < 0.01.

Fig. 8.

Effect of the PKC inhibitor Ro31-8220 on AMPH- and K+-mediated DA release in striatal slices from saline (A)- and AMPH (B)-pretreated rats. Striatal slices were incubated with 1 μm Ro31-8220 for 30 min before being given a 2.5 min bolus of KRB, 1 μm Ro31-8220 (Ro), 1 μm AMPH (A), 1 μm AMPH + 1 μm Ro31-8220 (A+Ro), 50 mm KCl (K), or 50 mm KCl + 1 μm Ro31-8220 (K+Ro). Results are presented as picomoles of DA per milligram wet weight (ww) ± SEM of the peak fraction containing maximal DA (n = 3). A, Effect of Ro31-8220 on DA release in saline-pretreated rats. ANOVA for AMPH-mediated DA release and controls, p < 0.0001.a, In post hoc Tukey–Kramer tests,A differs from KRB, Ro, and A+Ro samples at p < 0.01. ANOVA for K+-mediated DA release and controls,p < 0.0001. b, Kdiffers from KRB and Ro samples atp < 0.001. B, Effect of Ro31-8220 on DA release in AMPH-pretreated rats. ANOVA for AMPH-mediated DA release and controls, p < 0.0008.a, In post hoc Tukey–Kramer tests,A differs from KRB and Roat p < 0.01 and from A+Ro atp < 0.05. ANOVA for K+-mediated DA release and controls, p < 0.0003.b, K differs from KRB andRo at p < 0.001 and fromK+Ro at p < 0.01.KRB differs from K+Ro atp < 0.05. c, In comparison of saline pretreatment (A) versus AMPH pretreatment (B), saline–AMPH samples differ from AMPH–AMPH samples at p < 0.02 by Student’s ttest. d, Saline–K+ samples differ from AMPH–K+ samples at p < 0.01 by Student’s t test.