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 Rats were pretreated for 5 d with 2.5 mg/kg amphetamine or saline and withdrawn from drug for 10 d. The selective CaM kinase II inhibitor KN-93 (1 μm), but not the inactive analog KN-92, attenuated the Ca2+-dependent amphetamine-mediated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 mmKCl 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 μm 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.
The catecholamine dopamine (DA) can be released from terminals and dendrites by exocytosis, a Ca2+- and synaptic vesicle-dependent process, or by a Ca2+-independent, plasmalemma transporter-mediated mechanism that is used by the psychostimulant amphetamines (AMPHs) (Raiteri et al., 1979). Many behavioral effects elicited by AMPH are attributed to its ability to release DA from presynaptic nerve terminals and to inhibit the reuptake of DA from the synapse (Seiden et al., 1993). DA release after acute administration of AMPH is Ca2+ independent; it relies on cytosolic DA and vesicular DA that permeates into the neuronal cytoplasm from synaptic vesicles (Chiueh and Moore, 1975; Raiteri et al., 1979; Jones et al., 1998). Repeated, intermittent treatment of rats with AMPH or cocaine leads to an enhanced release of DA from areas such as the striatum and nucleus accumbens in response to stimuli such as AMPH or depolarization (Robinson and Becker, 1982; Robinson et al., 1985; Castañeda et al., 1988; Kolta et al., 1989; Wolf et al., 1993; Pierce and Kalivas, 1997a). The enhanced component of the AMPH-induced DA release after repeated AMPH or cocaine is distinguished from that released in nontreated rats by its Ca2+ dependency (Warburton et al., 1996; Iwata et al., 1997; Pierce and Kalivas, 1997a). We have identified additional Ca2+-related activities in rat striatum that develop after repeated, intermittent AMPH. After cessation of repeated AMPH, we found an increase in the Ca2+-binding protein calmodulin (CaM) and in the activity of Ca2+- and CaM-dependent protein kinase II (CaM kinase II) in striatal synaptosomes as well as an increase in the phosphorylated state of the vesicular protein synapsin I at the CaM kinase II substrate site (Iwata et al., 1996, 1997). Both CaM kinase II and synapsin I are involved in neurotransmitter release; CaM kinase II enhanced Ca2+-dependent glutamate release from nerve endings purportedly via an enhanced phosphorylation of synapsin I (Llinas et al., 1991; Nichols et al., 1992). Inhibitors of CaM kinase II blocked a Ca2+-dependent component of amphetamine-mediated DA release in the nucleus accumbens of rats treated with repeated cocaine (Pierce and Kalivas, 1997a) and the sensitized locomotor behavior (Pierce et al., 1998).
The goal of the present study was to examine the role of CaM kinase II in the Ca2+-dependent component of DA release in rat striatum after repeated AMPH in response to a challenge with AMPH. We also investigated whether the enhanced depolarization-mediated DA release after repeated, intermittent AMPH was dependent on CaM kinase II. The source of the releasable pool of DA affected by an AMPH challenge after a repeated AMPH treatment is unknown. Synaptic vesicles could be integral to DA release by both stimuli because DA release in response to both depolarization and AMPH is enhanced after repeated AMPH. To determine whether synaptic vesicles were a source of DA for the enhanced DA release after repeated AMPH, we examined whether reserpine treatment, which depletes vesicular DA, would abolish the amphetamine-mediated Ca2+-dependent DA release elicited by the repeated AMPH.
MATERIALS AND METHODS
Repeated AMPH regimen. Female Holtzman rats (179–190 gm; Harlan Sprague Dawley, Indianapolis, IN) were given intraperitoneal injections of AMPH (2.5 mg/kg) or saline once a day for 5 d. Saline- and AMPH-pretreated rats were killed 10 d after the last injection. This treatment was chosen because it produces a behavioral sensitization in response to a challenge dose of AMPH (Robinson and Becker, 1982).
Striatal slice preparation. Rats were killed by decapitation, and the striatum was removed and dissected on ice using a brain-cutting block as described (Heffner et al., 1980). The striatal tissue of each rat was divided into 1 mm3 pieces and placed immediately into ice-cold Krebs’ Ringer buffer (KRB) containing 125 mm NaCl, 2.7 mm KCl, 1 mmMgCl2, 1.2 mm CaCl2, 1.2 mm KH2PO4, 10 mm glucose, 24.9 mm NaHCO3, 0.25 mm ascorbic acid, and 10 μm pargyline. The buffer was oxygenated with 95% O2/5% CO2 for 1 hr, with pH at 7.4.
DA release assay. Striatal slices were weighed and placed into a designated chamber of a Brandel SF-12 superfusion apparatus (Gaithersburg, MD) onto a Whatman GF/B glass filter (Maidstone, UK). Superfusion chambers were maintained at 37°C, and medium was perfused through the chambers at 100 μl/min. Samples were collected at 5 min intervals. All chambers were perfused with KRB or drug for 30 min, followed by a 2.5 min bolus of the following drug combinations: 1 μm AMPH, 1 μm AMPH + 10 μmKN-93, 10 μm KN-93 alone, and KRB as control. The bolus of AMPH was given at fraction 7. Analysis of the time to travel through the tubing demonstrated that the bolus would arrive at the tissue at fraction 9. In some experiments, KN-92 was substituted for KN-93. Experiments measuring depolarization contained 50 mm KCl or 50 mm KCl + 1 μm KN-93. In these experiments, Na+ was commensurately lowered in the KRB to maintain osmolarity. The bolus drug combinations were replaced with fresh KRB or with KN-93 and/or KN-92, and sample collection continued for 40 more minutes. Samples were collected into vials containing 25 μl of internal standard solution [0.05N HClO4, 4.55 mm dihydroxybenzylamine (DHBA), 1 m metabisulfate, and 0.1 m EDTA]. Samples were stored at −70°C until analysis. The DA content of the samples was analyzed by HPLC with electrochemical detection using DHBA as an internal standard (Becker et al., 1984). Statistical significance was determined using one-way ANOVA with a post-test Tukey–Kramer multiple comparison analysis or by Student’s t test.
Reserpine treatment and total DA measurement. Female Holtzman rats were pretreated with repeated AMPH or saline as discussed above. On day 9 after the last dose of drug, rats were injected subcutaneously with 5 mg/kg reserpine or vehicle (1.5% glacial acetic acid) and killed 18 hr later. DA was measured in each striatum to determine the degree of depletion and was, as we found previously (Kantor and Gnegy, 1998), depleted 95% in both saline- and AMPH-pretreated rats.
[3H]DA uptake. Striatal slices from AMPH- and saline-pretreated rats were weighed, placed into vials containing KRB with and without 1.2 mm CaCl2 at 37°C for 5 min, and incubated for 20 more minutes in the presence or absence of 10 μm KN-93, 10 μm nomifensine, or 5 μm GBR-12935. After preincubation, [3H]DA (18.3 Ci/mmol) was added to a concentration of 33 nm, and the incubation proceeded for an additional 5 min. The reaction was terminated by the addition of 10 ml of ice-cold saline followed by filtration on GF/C filters (Whatman) and two more saline washes. Filters were counted in ScintiVerse BD in a Beckman LS 5800 scintillation counter. Data were adjusted for the weight of the slices.
Drugs. AMPH was obtained from the University of Michigan Laboratory of Animal Medicine (Ann Arbor, MI). HPLC-grade chemicals were purchased from American Bioanalytical (Natick, MA). KN-93, KN-92, KN-62, and GBR-12935 were purchased from Calbiochem (La Jolla, CA), and reserpine was obtained from Research Biochemicals (Natick, MA). Nomifensine was the generous gift of Dr. James Woods (Department of Pharmacology, University of Michigan, Ann Arbor, MI).
Effect of AMPH challenge on DA release from pretreated rats
The endogenous DA released in response to 1 μm AMPH was measured in striatal slices 10 d after the last pretreatment dose of saline or AMPH (2.5 mg/kg per day for 5 d). As shown in Figure 1, AMPH-mediated DA release from striatal slices was twofold greater from AMPH-pretreated rats than from saline-pretreated rats. Baseline values did not differ.
Effect of KN-93 and KN-92 on AMPH-mediated DA release in pretreated rats
The effect of the selective CaM kinase II inhibitor KN-93 and its inactive analog KN-92 on AMPH-induced DA release in striatal slices from saline- and AMPH-pretreated rats was examined. At 10 μm, KN-93 had no effect on either basal DA levels or AMPH-mediated DA release in saline-pretreated rats (Fig.2 A). KN-93, however, did significantly inhibit AMPH-induced DA release in AMPH-pretreated rats (Fig. 2 B). The degree of inhibition was ∼50%; the amount of remaining DA released was equal to the level of AMPH-induced DA release in slices from saline-pretreated rats. At 10 μm, KN-92, the inactive analog of KN-93, had no effect on basal levels or DA released by 1 μm AMPH in either AMPH- or saline-pretreated rats (Fig. 2 C,D). The same results were seen when striatal tissues were pretreated with a different inhibitor of CaM kinase II, KN-62. In striatal slices from saline-pretreated rats, values for peak DA released in response to 1 μm AMPH in the absence and presence of 5 μmKN-62 are 0.34 and 0.30 pmol/mg wet weight, respectively. In AMPH-pretreated rats, values for DA released in response to 1 μm AMPH in the absence and presence of 5 μmKN-62 are 0.93 and 0.44 pmol/mg wet weight, respectively. Values are the average from two experiments whose values did not differ >10%.
Depolarization-mediated DA release and the effect of KN-93 after repeated AMPH
Castañeda et al. (1988) demonstrated that electrical- and depolarization-mediated DA release was enhanced in rats treated with a sensitizing regimen of repeated AMPH that differed from this regimen. To ensure that there was enhanced depolarization-mediated DA release in our regimen, we perfused striatal slices from saline- and AMPH-pretreated rats with 50 mm KCl. As shown in Figure3, depolarization-mediated DA release was increased twofold after withdrawal from the repeated AMPH regimen compared with that in saline controls. However, the K+-mediated DA release was not altered by KN-93 (Fig.4 A,B) or KN-92 (data not shown) alone in striatal slices from either saline- or AMPH-pretreated rats.
Effect of the CaM kinase II inhibitor on [3H]DA uptake
Because KN-93 could be inhibiting AMPH action by blocking its uptake, we assessed its effect on the uptake of [3H]DA in striatal slices from rats pretreated with saline or AMPH. Experiments were performed in the presence and absence of Ca2+ to determine whether there was a Ca2+-dependent component of [3H]DA uptake after repeated AMPH. In contrast to the Ca2+ dependency of transporter-mediated DA release after repeated AMPH, striatal DA uptake was Ca2+ independent in both AMPH- and saline-pretreated rats (Fig. 5). KN-93 did not significantly affect DA uptake in the striatum from either saline- or AMPH-pretreated rats. Nomifensine and GBR-12935, which are dopamine transporter blockers, inhibited [3H]DA uptake into striatal slices equally well from either AMPH- and saline-pretreated animals.
Effect of reserpine on AMPH-mediated DA release after repeated AMPH
Although KN-93 had no effect on depolarization-mediated DA release, the Ca2+ dependency of both the enhanced AMPH-mediated and the depolarization-mediated DA release suggested that synaptic vesicles could be involved in the increased response to both stimuli. To test for a requirement for synaptic vesicles in the enhanced stimulus-induced DA release after repeated AMPH, we pretreated rats with saline or AMPH and gave the rats reserpine (5 mg/kg, s.c.) or vehicle 9 d after the last treatment. There were thus four groups of rats differing in repeated treatment and injection type on day 9: saline-pretreated rats given vehicle on day 9 (Fig.6, S), saline-pretreated rats given reserpine on day 9 (rS), AMPH-pretreated rats given vehicle on day 9 (A), and AMPH-pretreated rats given reserpine on day 9 (rA). Rats were killed the next day, which was day 10 after the last drug injection, and DA release in response to either AMPH or K+ was measured in the striatal slices. The amount of DA released in response to 1 μm AMPH is shown in Figure 6 A. Reserpine treatment on day 9 had no effect on endogenous DA release in response to 1 μm AMPH in saline-pretreated rats, as we have seen previously (Kantor and Gnegy, 1998). As shown in Figure6 A, reserpine given on day 9 similarly had no significant effect on AMPH-mediated DA release in AMPH-pretreated rats. The controls (KRB alone in the perfusion) for all groups gave the same values and were treated as one line on Figure6 A to avoid visual clutter. The effect of reserpine on K+-mediated DA release after repeated saline or AMPH is shown in Figure 6 B. As expected, reserpine pretreatment abolished K+-mediated DA release in slices from rats repeatedly treated with either saline or AMPH.
Effect of reserpine treatment on KN-93 inhibition of AMPH-mediated DA release in pretreated rats
To help delineate the site of action of KN-93 in inhibiting striatal AMPH-mediated DA release in AMPH-pretreated rats, we examined the effect of reserpine on the KN-93 inhibitory action. Because the vesicles were obviously depleted by reserpine (Fig.6 B), a lack of effect of KN-93 in reserpine-pretreated rats would suggest that the drug was acting on CaM kinase II on the synaptic vesicles. As shown in Figure2 A, 10 μm KN-93 had no effect on AMPH-mediated DA release from saline-pretreated rats. Reserpine given on day 9 did not alter that result (Fig.7 A). Similarly, the ability of 10 μm KN-93 to inhibit the enhanced component of AMPH-mediated DA release in AMPH-pretreated rats was unaltered by reserpine given on day 9 (Fig. 7 B). KN-93 (10 μm) did not affect basal DA levels under any treatment condition.
Protein kinase C inhibitors attenuated AMPH- but not K+-stimulated DA release in saline- and AMPH-pretreated rats
We have shown previously that inhibitors of protein kinase C (PKC) completely inhibit AMPH-mediated DA release in the rat striatum and nucleus accumbens independently of extracellular calcium (Kantor and Gnegy, 1998; Browman et al., 1999). The specificity of the CaM kinase II inhibition of AMPH-mediated DA release was examined by determining whether PKC inhibition attenuated DA release mediated by AMPH or K+ in AMPH-pretreated rats. Striatal slices from rats pretreated with saline or AMPH were perfused with 1 μm Ro31-8220 for 30 min before the bolus of AMPH or KCl. As shown in Figure8 A, Ro31-8220 treatment completely blocked AMPH-mediated DA release in striatal slices from saline-pretreated rats but had only a partial effect in AMPH-pretreated rats (Fig. 8 B). On the contrary, Ro31-8220 perfusion had no effect on K+-stimulated DA release in either saline- or AMPH-pretreated rats. There appeared to be a small degree of inhibition in AMPH-pretreated rats, but there was no significant difference between K+-stimulated values in the absence or presence of Ro31-8220. When calculated as fold stimulation over control, the values in AMPH-pretreated rats for K+ and K+ + Ro31-8220 samples were 5.9 ± 1.0 and 5.1 ± 1.2, respectively, again showing no significant difference.
Our results demonstrate that, after reexposure to AMPH, the striatum from rats treated repeatedly with AMPH displayed a DA release that was twofold higher than that from saline-treated rats. The increase in released DA was Ca2+ dependent, and the involvement of a Ca2+- and CaM-dependent protein phosphorylation was demonstrated by the ability of KN-93 to inhibit the Ca2+-dependent component. Functioning synaptic vesicles seemed not to be integral to the enhanced AMPH-mediated DA release. The twofold enhancement in K+-induced DA release after repeated AMPH, although Ca2+ and vesicular dependent, differed from the AMPH-mediated release in that it was unaffected by KN-93. Similarly, there is a component of PKC dependence in the AMPH-mediated DA release that is not reflected in K+-stimulated DA release. The results concerning AMPH-mediated DA release correspond to those of Pierce and Kalivas (1997a) who, using microdialysis, demonstrated a Ca2+-dependent enhancement in AMPH-induced DA release in the nucleus accumbens of rats treated repeatedly with cocaine. Ca2+ entry into the terminal and activation of Ca2+- and CaM-dependent protein kinase activity were crucial to the enhanced AMPH-induced DA release, but exocytosis was not. Consequently, the enhancement in AMPH-induced DA release after repeated AMPH or cocaine may involve similar mechanisms.
Vesicular involvement in enhanced AMPH-mediated DA release
It has been postulated that the enhancement in AMPH-induced release in cocaine- or AMPH-pretreated rats could be caused by a redistribution of DA-containing synaptic vesicles that might position them more closely in apposition with plasmalemmal DA transporters (Robinson, 1991; Pierce and Kalivas, 1997b). “Leakage” or increased transport of DA from the vesicle could serve as the source of the enhanced DA. In support of that hypothesis, we found that repeated AMPH leads to a long-lasting elevation in site 3 phosphosynapsin I in rat striatum (Iwata et al., 1996, 1997). Dephosphosynapsin I mediates the formation of ternary complexes with synaptic vesicle and actin filaments. Phosphorylation of synapsin I at sites 2 and 3, substrate sites for CaM kinase II, abolishes those interactions. Because synapsin I is involved in maintaining a clustered, reserve pool of synaptic vesicles used during high synaptic activity (Pieribone et al., 1995), it is conceivable that disruption of this pool could lead to a wider distribution of vesicles that could enhance the supply of DA to be released in response to AMPH. Another role for vesicles could involve a direct interaction of AMPH with the vesicular monoamine transporter VMAT2 that could increase the availability of DA at the plasmalemma transporter. Because CaM kinase II is located on vesicles, as well as in the cytoplasm (Dunkley et al., 1988; Benfenati et al., 1992), vesicular CaM kinase II could be altering AMPH action at the vesicles.
Our studies, however, suggest that synaptic vesicles do not provide the source of DA for the enhanced AMPH response after repeated AMPH. Reserpine pretreatment completely abolished K+-stimulated DA release but did not affect AMPH-stimulated DA release in the striatum of rats pretreated with either saline or AMPH. In addition, reserpine pretreatment did not alter the ability of KN-93 to inhibit the Ca2+-dependent enhancement in AMPH-induced DA release. Although it is possible that the reserpine treatment did not deplete all of the vesicles, one might expect that the responses would have been diminished if vesicles were strongly involved in these effects. Reserpine completely ablated [3H]tetrabenazine binding to VMAT2 in rat striatum 1 d after injection, suggesting that vesicles are not functional (Naudon et al., 1995). Therefore, the synaptic vesicles do not seem to be the source of the increased DA released by AMPH through the transporter. The possibility that a vesicular CaM kinase II or a vesicular substrate for the enzyme is important for the process independent of active VMAT2 cannot be discounted, however. Our conclusion is predicated on the use of a relative low dose of AMPH, 1 μm, as our challenge dose. There is increasing vesicular involvement in AMPH-mediated DA release as the dose of AMPH is increased (Seiden et al., 1993). Had we used higher doses of AMPH, such as 100 μm, at which vesicles play a prominent role in the amount of DA released (Sulzer et al., 1995), no doubt a vesicular involvement would have been detected. However, we have demonstrated a sensitized DA release in response to 1 μmAMPH that remained unaltered by pretreatment with reserpine. Therefore the enhanced AMPH-mediated DA release after repeated AMPH is not dependent on functioning synaptic vesicles.
Role of CaM kinase II in enhanced AMPH-mediated DA release
The most parsimonious conclusion to explain the enhanced AMPH-induced DA release is that intraterminal CaM kinase II activity is maintaining phosphorylation of a substrate that enhances outward transport. We demonstrated that CaM kinase II activity is enhanced in striatal synaptosomes after repeated AMPH and that enhanced AMPH-induced DA release can be demonstrated in the synaptosomes (Iwata et al., 1997). The CaM kinase II activity is likely ongoing because the inhibitor KN-93 was readily effective. The nature of the substrate is unknown. It could be an ion channel or cytoskeletal protein affecting transporter function, or it could be the plasmalemmal DA transporter itself. The DA transporter from both rat and human contains a consensus sequence for CaM kinase II (Giros and Caron, 1993). Whatever the substrate, the modification is such that it affects primarily outward-directed transporter function. Because there is no change in basal DA release, the modification is unlikely to involve a global alteration in ion gradients such as that an inhibition of Na+,K+-ATPase would provide but could involve an AMPH-mediated effect on an ion gradient. Interestingly, ouabain-stimulated DA release from rat striatum is also enhanced after repeated AMPH (Kanzaki et al., 1992). The modification leading to the enhanced transporter-mediated DA release did not alter all transporter functions. We and others (Kuczenski and Segal, 1988) have found no change in DA uptake after repeated AMPH, showing that inward transport was unaltered. Ca2+ did not affect uptake in either saline- or AMPH-pretreated rats. This is in contrast to the report of Uchikawa et al. (1995) who found that Ca2+ and CaM increased [3H]DA uptake in naive rats. We also found that neither Ca2+ (data not shown) nor repeated AMPH altered the ability of maximal concentrations of the uptake blockers nomifensine and GBR-12935 to inhibit DA uptake. In microdialysis studies, Pierce and Kalivas (1997a) reported that levels of extraterminal DA in response to GBR-12909 were enhanced after repeated cocaine. This could be caused by the increase in exocytotic, impulse-dependent DA release occurring after repeated AMPH rather than by an altered effect of GBR-12909 on the DA transporter.
It is possible for a modification to affect outward transport through the plasmalemmal DA transporter without substantively altering inward transport. We found that selective inhibitors of protein kinase C blocked AMPH-mediated DA release in the striatum and nucleus accumbens from naive animals (Kantor and Gnegy, 1998; Browman et al., 1999) but slightly increased [3H]DA uptake (Kantor and Gnegy, 1998). Although the selective PKC inhibitor Ro31–8220 completely blocked AMPH-mediated DA release from naive (Kantor and Gnegy, 1998; Browman et al., 1999) and saline-pretreated rats (this study), this inhibitor only partially attenuated AMPH-mediated DA release from AMPH-pretreated rats. Inhibition of AMPH-induced DA release by the PKC inhibitors is entirely independent of extracellular calcium (Kantor and Gnegy, 1998). Thus the Ca2+-sensitive and -insensitive phases of AMPH-induced DA release in AMPH-pretreated rats seem to be separable and controlled by different protein kinase mechanisms.
Enhanced K+-mediated DA release
Repeated AMPH also led to an increase in depolarization-mediated, vesicular-dependent exocytotic release (this study) (Castañeda et al., 1988). Possible explanations for an increase in K+-evoked DA release are (1) movement of vesicles from a reserve to a more readily releasable pool, (2) an increase in the number of vesicles, (3) alteration in a plasmalemmal or vesicular membrane protein that heightens exocytosis, or (4) altered Ca2+ channel activity leading to increased intracellular Ca2+. The increase we reported in site 3 phosphosynapsin I (Iwata et al., 1996) could conceivably contribute to an increase in the readily releasable vesicular pool because it could decrease the attachment of vesicles to a confining cytoskeleton. On the other hand, KN-93 did not block K+-stimulated DA release, suggesting that ongoing Ca2+ and CaM protein phosphorylation does not modulate the depolarization-enhanced release. It is unlikely that the total number of vesicles increased because we found no change in total synapsin I (Iwata et al., 1996), which seems to be a component of every vesicle, including dopaminergic vesicles, in the striatum (Greengard et al., 1994). Although the increase in site 3 phosphosynapsin I could contribute to increased exocytotic release of DA, it is unclear whether the increase is in dopaminergic vesicles. A more probable option is that there is an increase in Ca2+ availability through Ca2+ channels that could lead to increased exocytotic release. Our results further suggest that ongoing PKC activity is not instrumental in mediating enhanced K+-induced DA release after repeated AMPH.
We have demonstrated that repeated AMPH treatment leads to an increase in DA released in response to AMPH and K+depolarization. Although the increased DA released in response to both stimuli was Ca2+ dependent, our experiments suggested that a Ca2+- and CaM-dependent phosphorylation was critical for the enhanced release by AMPH but not for that released by depolarization. On the contrary, the depolarization-mediated DA release was entirely vesicular, whereas vesicles were not required for the AMPH-mediated release. The results suggest that a CaM-dependent phosphorylation of a protein, perhaps the plasmalemma DA transporter itself or a membrane protein affecting the transporter function, could be responsible for the enhanced AMPH-induced release.
This work was supported by the National Institute on Drug Abuse (NIDA) Grant DA-05066 and NIDA Interdisciplinary Training Grant DA-07267 at the University of Michigan Substance Abuse Research Center (L.K.). We thank Terry Robinson for the use of his HPLC and for helpful conversations.
Correspondence should be addressed to Dr. Margaret E. Gnegy, Department of Pharmacology, 2220E MSRB III, University of Michigan Medical School, Ann Arbor, MI 48109-0634.