Introduction

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-Receptors were first identified in 1976 by Martin et al. (1976) as the sites through which the psychotomimetic effects of SKF10,047 were mediated. Since that time, progress in identifying their physiological function(s) has been relatively slow compared with other receptor systems. There are at least two types of -receptors, designated ₁ and ₂ (Hellewell and Bowen, 1990; Quirion et al., 1992). -Receptors are distributed widely in the periphery and central nervous system (Walker et al., 1990). In brain, they are localized especially in motor and limbic systems (Gundlach et al., 1986), areas typically subserved by catecholaminergic neurotransmission. ₁- and ₂-receptors are colocalized in most regions, and have been reported to be present in a ratio of 4.3:1 in striatum (Bouchard and Quirion, 1997). ₁-Receptors were cloned first from guinea pig liver by Hanner et al. (1996) and the corresponding human gene further characterized by Prasad et al. (1998).

₁-Receptors have been associated with second messenger activation and inhibition in several model systems. There is disagreement whether ₁-receptors are coupled through G proteins. Early binding studies showed changes in binding parameters in the presence of GTP or nonhydrolyzable analogs (Beart et al., 1989; Itzhak, 1989), but the cloning of the ₁-receptor protein revealed a structure too small to be a typical seven transmembrane-spanning entity common to other G protein-coupled receptors. In our laboratory, we have not found evidence of direct coupling to G proteins (Hong and Werling, 2000, 2001), but have not ruled out an indirect coupling. ₁-Receptors have been associated with calcium homeostasis (Brent et al., 1997; Klette et al., 1997; Hayashi et al., 2000).

To date, the ₂-receptor has not been cloned, and fewer reports of associated signaling mechanisms have appeared. Vilner and Bowen (2000) have demonstrated a regulation of stimulation of Ca²⁺ release from ER stores by ligands with affinity at ₂-receptors, and a reversal of that increase in intracellular calcium by two -receptor antagonists. ₂-Receptors are likely to be located intracellularly. This feature affects access to the receptors by ligands dependent upon characteristics of the ligand, such as lipophilicity, and characteristics of the assays system, such as pH, important to the observed physiological response (for review, see Bowen, 2000).

In this laboratory, we have studied modulation of catecholamine release by  receptor ligands acting through ₁- and ₂-receptors (Gonzalez-Alvear and Werling, 1994, 1995; Izenwasser et al., 1998; Weatherspoon and Werling, 1999). The -selective agonist (+)-pentazocine has a K_i for binding to ₂-receptor sites that is reported to be as low as 500 nM (Quirion et al., 1992) and as high as 1.5 µM (Vilner and Bowen, 1992). We have demonstrated that (+)-pentazocine, at concentrations consistent with ₂-receptor activation, enhances amphetamine (AMPH)-stimulated [³H]dopamine (DA) release in striatal slices (Izenwasser et al., 1998) and PC12 cells (Weatherspoon and Werling, 1999). In both model systems, the enhancement is blocked by selective ₂-receptor antagonists and is dependent on Ca²⁺. Whereas inclusion of EGTA did not affect stimulation of [³H]DA by amphetamine, it completely abolished the enhancement of release by pentazocine acting via the ₂-receptor. In PC12 cells, we found the enhancement of AMPH-stimulated release also to be dependent on the activity of Ca²⁺/calmodulin kinase II (Ca²⁺/CaM kinase II); in the presence of Ca²⁺/CaM kinase II inhibitors, no enhancement was observed.

In the current study, we sought to identify second messenger systems underlying the regulation of DA transporter (DAT) activity by ₂-receptors in brain tissue. We tested drugs with antagonist activity at Ca²⁺ channels, as well as thapsigargin, which depletes intracellular Ca²⁺ stores, and Ca²⁺/CaM kinase II, as well as activators and inhibitors of protein kinase C (PKC) to learn whether any of these could mimic or inhibit the effects of (+)-pentazocine on AMPH-stimulated DA release.

	Materials and Methods

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All experiments were done in accordance with the guidelines and under approval of The George Washington University Animal Use and Care Committee.

Preparation of Synaptosomes from Brain Tissue and Measurement of [³H]DA Uptake into Brain Synaptosomes. Sprague-Dawley male rats were euthanized by decapitation, the brains removed to ice, and the striata dissected. The tissue was weighed and homogenized in 10 volumes of 0.32 M sucrose/5 mM HEPES. The homogenate was then centrifuged for 10 min at 1000g and the supernatant collected and kept on ice (SUP1). The pellet (P1) was rehomogenized in the same volume 0.32 M sucrose/5 mM HEPES. This new homogenate was recentrifuged for 10 min at 1000g. The supernatant (SUP2) was collected and the pellet discarded. SUP1 and 2 were combined and centrifuged for 20 min at 20,000g. The supernatant was discarded. The upper, buffy coat containing the synaptosome fraction was separated from the lower, brown mitochondrial layer by gentle agitation into 5 ml of modified Krebs-HEPES buffer (MKB; 127 mM NaCl, 5 mM KCl, 1.3 mM NaH₂PO₄, 1.2 mM MgSO₄, 2.5 mM CaCl₂, 15 mM HEPES acid, 10 mM dextrose, pH adjusted to 7.4 with NaOH). The synaptosomes were washed three times in MKB with centrifugations at 20,000g for 10 min between each wash. MKB (5 ml) was added to the final synaptosomal pellet and homogenized. MKB (10 ml) was added for a final volume of 15 ml. To this suspension 1 mM ascorbic acid was added. One milliliter of the suspension was aliquoted into tubes and (+)-pentazocine added at appropriate concentrations (Table 1). To each tube [³H]DA was added to begin the assay. The tubes were incubated in a water bath at 37°C for 30 min. The assay was terminated by addition of 2 ml of ice-cold MKB, and the samples collected on Whatman (Maidstone, UK) 2.4-cm GF/B glass filter disks (previously soaked in 0.1% polyethyleneimine for at least 1 h) over a vacuum harvester. The filters were then transferred into individual 20-ml scintillation vials to which 1 ml of methanol and 2 ml of 0.2 N HCl were added. The vials were then capped and placed in a 70°C oven for 2 h. After cooling, scintillation cocktail was added and the vials were left for 24 h. The next day, total accumulated radioactivity was determined by liquid scintillation spectroscopy. Data were expressed as a percentage of the radioactivity accumulated by control (percentage of control uptake). Data were analyzed using one-way analysis of variance (ANOVA).

Measurement of Stimulated [³H]DA Release from Brain Slices. Sprague-Dawley male rats (300-500 g) were euthanized by decapitation, the brains removed to ice, and the striata dissected. The tissue was weighed and chopped into 250- × 250-µm slices on a Sorvall TC2 tissue slicer with two successive cuts at an angle of 90°. The tissue slices were then suspended in 20 ml of oxygenated MKB (127 mM NaCl, 5 mM KCl, 1.3 mM NaH₂PO₄, 1.2 mM MgSO₄, 15 mM HEPES acid, 10 mM dextrose, pH adjusted to 7.4 with NaOH). Ca²⁺ was omitted from the buffer when using AMPH as a stimulus to prevent any exocytotic release. MKB buffers were saturated with 95% O₂, 5% CO₂ and warmed to 37°C throughout the experiment. Three washes in approximately 20 ml of MKB were performed. After the third wash, the tissue suspensions were incubated in 20 ml of MKB containing 15 nM [³H]DA and 0.1 mM ascorbic acid at 37°C for 30 min. Tissue preparations were washed twice (5 min/wash) in 20 ml of MKB and once in MKB containing domperidone, which was included in all subsequent steps, to prevent feedback inhibition of release through presynaptic dopamine receptors. When thapsigargin was used to deplete ER Ca²⁺ stores, it was added during the final wash. Tissue preparations were then suspended one final time in a volume of 7.5 ml of MKB and distributed in 275-µl aliquots between glass fiber filter discs into chambers of a Brandel Inc. (Gaithersburg, MD) superfusion apparatus.

Chemicals and Reagents. Drugs and reagents were kindly provided by or obtained from the following sources: amphetamine, domperidone, GF109,203X, KN-62, KN-92, KN-93, nitrendipine (NIT), and phorbol-12-myristate-13-acetate (PMA; Sigma/RBI, Natick, MA); TTX, -conotoxin, thapsigargin, and 4-PMA (Sigma-Aldrich, St. Louis, MO); [³H]DA (specific activity 46-51 Ci/mmol) (Amersham Biosciences, Inc., Piscataway, NJ); (+)-pentazocine (Research Technology Branch, National Institute on Drug Abuse, Rockville, MD); and endo-N-(8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-2,3-dihydro-(1-methyl)ethyl-2-oxo-1H-bezimidazole-1-carboxamide hydrochloride (BIMU-8; Dr. Doug Bonhaus, Roche Bioscience, Palo Alto, CA).

	Results

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Figure 1 is a representative result from a release experiment showing a time course for release produced by amphetamine alone in both stimulus intervals and amphetamine alone in the first stimulus interval, and combined with (+)-pentazocine in the second interval. As we have observed in our previous experiments, (+)-pentazocine enhanced amphetamine-stimulated release (Figs. 1 and 2). For clarity, in Fig. 1 we show data from a single tissue sample for each of two treatments, but in our experiments we always include triplicate determinations for each treatment. The K_i for (+)-pentazocine binding to ₂-receptor sites is between 500 nM (Quirion et al., 1992) and 1.5 µM (Vilner and Bowen, 1992). The concentration of (+)-pentazocine used was 1 µM chosen to be near the K_i for (+)-pentazocine at the ₂-receptor. This should produce an occupancy of 67% if the K_i is closer to the 500 nM value (Quirion et al., 1992) and 47% if the K_i value is 1.5 µM (Vilner and Bowen, 1992) of ₂-receptors. This concentration has been shown to be on the ascending portion of a dose-response curve for inducing [³H]DA release (Izenwasser et al., 1998). The enhancement was reversed by the ₂-selective antagonist BIMU-8 (K_i for ₂, 20 nM; K_i for ₁, >1000 nM; Bonhaus et al., 1993) at 100 nM (Fig. 2). The only difference between previous experiments and the current experiments was the presence of TTX in the buffer in the current experiments (see Materials and Methods).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Representative result of a release experiment. Tissue is superfused for 30 min to establish baseline release before beginning of collection intervals (t = 0). The open circles represent results from a typical experiment in which amphetamine alone was introduced in both S1 and S2. The filled circles represent results from a typical experiment in which (+)-pentazocine was added into the ISI and S2. Typically, 10 µM amphetamine is added at t = 0, reaching tissue at arrow 1, to give the S1. (+)-Pentazocine and any potential antagonists are added to reach tissue at arrow 2, during the ISI so we can detect any potential effects on baseline release. Amphetamine (10 µM) is added again, reaching tissue at arrow 3, in the presence of (+)-pentazocine and/or other experimental drugs. The time between introduction of drug into the system and observed release (indicated by arrows 1, 2, and 3) is due to the time it takes for delivery of buffer containing drugs to travel through the tubing and reach the tissue. Time points included in calculation of release in S1, ISI, and S2 are indicated.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Effects of (+)-pentazocine on amphetamine-stimulated [3H]DA release. Striatal slices preincubated with 15 nM [3H]DA were stimulated with 10 µM AMPH alone, or in the presence of (+)-pentazocine (1 µM) or (+)-pentazocine and the selective 2-receptor antagonist BIMU-8 (100 nM). Data are expressed as percentage of release stimulated by AMPH alone (%control-stimulated release) in S2. Data were analyzed using one-way factorial ANOVA with post hoc Dunnett's tests. **, significantly different from control (p < 0.01). , significantly different from (+)-pentazocine (p < 0.05). N = 4 independent experiments, in which each treatment was tested in triplicate.

(+)-Pentazocine Does Not Affect Uptake of [³H]DA into Rat Striatal Synaptosomes. To determine whether the activation of ₂-receptors affects solely the outflow of [³H]DA or may also have effects on inward transport of [³H]DA, uptake assays on rat striatal synaptosomes were performed. Izenwasser et al. (1993) had previously shown that (+)-pentazocine did not inhibit uptake of [³H]DA into striatal slices at concentrations up to 10 µM. We tested uptake into synaptosomes to ensure there were no penetration problems or interneuronal effects contributing to results obtained in their experiments. If the enhancement of release by (+)-pentazocine was actually occurring as a result of inhibition of uptake, there would be less accumulation of [³H]DA into synaptosomes in the presence of (+)-pentazocine. Table 1 shows that (+)-pentazocine at concentrations ranging from 0.01 to 10 µM did not significantly affect the uptake of [³H]DA into synaptosomes. At concentrations of 0.1 and 1.0 mM, there is a slight suggestion of enhancement, but not inhibition, of uptake.

Ca²⁺/CaM Kinase II Inhibitors Do Not Affect (+)-Pentazocine Enhancement of Amphetamine-Stimulated [³H]DA Release. In a previous study from our laboratory (Weatherspoon and Werling, 1999), the Ca²⁺/CaM kinase II inhibitor KN-62 (K_i of 0.9 µM for Ca²⁺/CaM kinase II; Tokumitsu et al., 1990) abolished the (+)-pentazocine-mediated enhancement of amphetamine-stimulated [³H]DA release from PC12 cells. Therefore, in the current study, we first investigated the potential role of the Ca²⁺/CaM kinase II in the ₂ signaling pathway in rat striatal slices (Fig. 3). Treatment with 1 µM (+)-pentazocine during S2 provoked the usual enhancement of 10 µM amphetamine stimulation of [³H]DA release. However, treatment of striatal slices in S2 with 10 µM KN62, which would be expected to completely inhibit Ca²⁺/CaM kinase II, showed no significant reduction of the (+)-pentazocine enhancement. KN-62 by itself did not significantly affect the level of amphetamine-stimulated [³H]DA release. We also tested an alternate Ca²⁺/CaM kinase II inhibitor, KN-93 (K_i = 0.37 µM; Sumi et al., 1991) and its structural analog KN-92, which is devoid of Ca²⁺/CaM kinase II inhibition properties, as a negative control. Each was tested at 10 µM. Neither of these agents significantly reduced the enhancement by (+)-pentazocine. Values were as follows (compared with control amphetamine-stimulated release as 100%): amphetamine plus (+)-pentazocine, 154 ± 12% (N = 16); amphetamine plus (+)-pentazocine in the presence of KN-93, 127 ± 17% (N = 4); and amphetamine plus (+)-pentazocine in the presence of KN-92, 121 ± 7.7% (N = 4). A potential complicating factor in interpreting data on KN-93 is that it slightly, although not significantly, enhanced release stimulated by amphetamine alone to 120 ± 11%. In contrast, the inactive isomer KN-92 did not affect amphetamine-stimulated release (105 ± 7.7%)

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Effects of Ca2+/CaM kinase II inhibition on (+)-pentazocine enhancement of amphetamine-stimulated [3H]DA release. Striatal slices preincubated with 15 nM [3H]DA were stimulated with 10 µM AMPH alone, or in the presence of 1 µM (+)-pentazocine with or without 10 µM KN-62, as indicated. Data are expressed as percentage of release stimulated by AMPH alone (%control-stimulated release) in S2. Data were analyzed using one-way factorial ANOVA with post hoc Dunnett's tests. **, significantly different from control (p < 0.01). N  3 independent experiments, in which each treatment was tested in triplicate.

Role of Calcium in (+)-Pentazocine Enhancement of Amphetamine-Stimulated [³H]DA Release. We had previously found that enhancement of amphetamine-stimulated [³H]DA release by (+)-pentazocine was abolished by the inclusion of EGTA in both slices (Izenwasser et al., 1998) and PC12 cells (Weatherspoon and Werling, 1999). This suggested that even though no Ca²⁺ had been added to the buffer, there were sufficient Ca²⁺ stores within the striatal slices to support the enhancement of stimulated release by (+)-pentazocine. We investigated the potential role of entry of Ca²⁺ via L-type and N-type VDCCs. Inclusion of the L-type VDCC NIT (K_i for binding to L-type VDCC in rat brain is 0.34 nM; for review, see Godfraind et al., 1986) reduced release produced by amphetamine plus 1 µM (+)-pentazocine to a value that was not significantly different from enhancement stimulated by amphetamine alone (Fig. 4A). Nitrendipine alone seemed to show an enhancement of [³H]DA release; however, this enhancement was not significantly different from control levels. In contrast, the N-type VDCC inhibitor -conotoxin at 100 nM had no significant effect on enhancement by (+)-pentazocine. In matched experiments, amphetamine plus (+)-pentazocine produced 150 ± 6.5% (N = 16) compared with amphetamine alone as 100%, whereas amphetamine plus (+)-pentazocine in the presence of -conotoxin produced 130 ± 20% (N = 3).

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Effects of inhibition of L-type VDCC or ER calcium depletion on (+)-pentazocine-mediated enhancement of amphetamine-stimulated [3H]DA release. A, striatal slices preincubated with 15 nM [3H]DA were stimulated with 10 µM AMPH alone, or in the presence of 1 µM (+)-pentazocine with or without 100 nM NIT, as indicated. B, striatal slices loaded with 15 nM [3H]DA were preincubated for 5 min in the presence of 2 µM thapsigargin. The slices were then stimulated with 10 µM AMPH alone, or in the presence of 1 µM (+)-pentazocine. Data are expressed as percentage of release stimulated by AMPH alone (%control-stimulated release) in S2. Data were analyzed using one-way factorial ANOVA with post hoc Dunnett's tests. **, significantly different from control (p < 0.01). , not significantly different from control (p > 0.05). N  3 independent experiments, in which each treatment was tested in triplicate.

Role of PKC in (+)-Pentazocine-Mediated Enhancement of Amphetamine-Stimulated [³H]DA Release. We investigated the possible role of PKC as a second messenger system influenced by ₂-receptor activation (Figs. 5-7). GF 109,203X alone at 100 nM did not have any effect on amphetamine-stimulated [³H]DA release (Fig. 5A). Figure 5B shows the effects of increasing doses of the selective PKC inhibitor GF109,203X on the (+)-pentazocine-mediated enhancement. Increasing doses from 10 nM to 300 nM produced a dose-dependent inhibition of the (+)-pentazocine-mediated enhancement with an EC₅₀ of 190 nM, and with a maximum inhibition producing a value that was slightly below control amphetamine-stimulated release. At either a 100 or 300 nM concentration of GF 109,203X, release was not significantly different from control amphetamine-stimulated release. GF109,203X has an IC₅₀ value for PKC (undifferentiated for isozyme) of 20 nM and an IC₅₀ value for PKA of 2 µM (Toullec et al., 1991; Goldman et al., 1992). In subsequent time course studies, to selectively inhibit PKC, we used a concentration of 100 nM so that PKA would not be affected.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Effects of increasing concentrations of the selective PKC inhibitor GF109,203X on the (+)-pentazocine-mediated enhancement of amphetamine-stimulated [3H]DA release. A, striatal slices preincubated with 15 nM [3H]DA were stimulated with 10 µM AMPH alone, or in the presence of 1 µM (+)-pentazocine alone or 100 nM GF109,203X alone. B, striatal slices preincubated with 15 nM [3H]DA were stimulated with 10 µM AMPH in the presence of 1 µM (+)-pentazocine and GF109,203X at the concentrations indicated. Data are expressed as percentage of release stimulated by AMPH alone (%control-stimulated release) in S2. Data were analyzed using one-way factorial ANOVA with post hoc Dunnett's tests. **, significantly different from control (p < 0.05). , significantly different from (+)-pentazocine (p < 0.05). N  3 independent experiments, in which each treatment was tested in triplicate.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Effects of exposure of tissue to GF109,203X on the (+)-pentazocine-mediated enhancement of AMPH-stimulated [3H]DA release. Striatal slices were preincubated with 15 nM [3H]DA. A, tissue was stimulated with 10 µM AMPH alone or in the presence of 1 µM (+)-pentazocine. B, tissue was stimulated with 10 µM AMPH, 1 µM (+)-pentazocine, and 100 nM GF109,203X. GF109,203X (100 nM) was added before (t = 0, concomitant with introduction of S1), with (t = 6), or after (t = 10, concomitant with introduction of S2) introduction of (+)-pentazocine, as indicated. Data are expressed as percentage of release stimulated by AMPH alone (%control-stimulated release) in S2. Data are analyzed using one-way factorial ANOVA with post hoc Dunnett's tests. **, significantly different from control (p < 0.01). N = 7 independent experiments, in which each treatment was tested in triplicate.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Effects of PKC activation on (+)-pentazocine-mediated enhancement of amphetamine-stimulated [3H]DA release. Striatal slices preincubated with 15 nM [3H]DA were stimulated with 10 µM AMPH alone, or in the presence of 1 µM (+)-pentazocine and/or PMA or 10 µM 4-phorbol, as indicated. Data are expressed as percentage of release stimulated by AMPH alone (%control-stimulated release) in S2. Data were analyzed using one-way factorial ANOVA with post hoc Dunnett's tests. **, significantly different from control (p < 0.01). N  3 independent experiments, in which each treatment was tested in triplicate.

	Discussion

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The elucidation of the mechanisms underlying ₂-receptor activation and signal transduction are crucial to the understanding of ₂-receptor function. The goal of this study was to identify the signaling pathway(s) through which ₂-receptors regulate the outflow of [³H]DA via the DAT in slices of rat striatum.

Our data show that the activation of ₂-receptors enhances outward transport of [³H]DA. In our study with a striatal synaptosomal preparation, we have shown that (+)-pentazocine does not significantly affect inward transport (Table 1), although at 0.1 and 1.0 µM concentrations, the values are slightly higher than control. If the enhancement in release were due to inhibition of reuptake, there would have been less accumulation of [³H]DA in the presence of (+)-pentazocine. These data support the assertion that effects of (+)-pentazocine on amphetamine-stimulated release are mainly on release via the DAT, and not on uptake. These observations are in agreement with those of Izenwasser et al. (1993).

To our knowledge, the only previously identified signaling system associated with ₂-receptors has been regulation of intracellular Ca²⁺ levels as demonstrated by Vilner and Bowen (2000). They demonstrated that ₂-ligands produced an immediate, dose-dependent, and transient rise in intracellular Ca²⁺ originating from the ER that was blocked by the -receptor antagonists N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-dimethylamino)ethylamine (BD1047) and 1-[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine (BD1063). In addition, they observed a secondary sustained increase in intracellular Ca²⁺ upon prolonged exposure to ₂-agonists that was thapsigargin-insensitive.

The enhancement of amphetamine stimulated [³H]DA release via activation of ₂-receptors is not dependent on Ca²⁺ added to the buffer because we do not include Ca²⁺; however, our previous studies that show abolition of the (+)-pentazocine-mediated effect by inclusion of EGTA have suggested that it is dependent upon Ca²⁺ that derives from the tissue itself (Izenwasser et al., 1998; Weatherspoon and Werling, 1999). Because we are using a slice preparation, there is certainly Ca²⁺ contained within the preparation, either extra- or intracellularly. Our data are consistent with that of Vilner and Bowen (2000) in that a physiological response to ₂-receptor activation causes a change in intracellular Ca²⁺ levels. VDCCs regulate intracellular Ca²⁺ levels that can ultimately affect other signaling mechanisms, so they were analyzed in the context of ₂-receptor activation. The L-type VDCC inhibitor nitrendipine, but not the N-type inhibitor -conotoxin, reduced the (+)-pentazocine-mediated enhancement to levels that were not significantly different from control levels.

We also observed that prior depletion of ER Ca²⁺ stores with a 5-min exposure to 2 µM thapsigargin prevented (+)-pentazocine-mediated enhancement of amphetamine-stimulated release, suggesting that the endogenous stores required originated in the ER. This is consistent with the findings of Vilner and Bowen (2000) who identified a mobilization of ER Ca²⁺ stores by ₂-receptor agonists. It is possible that nitrendipine is able to block the (+)-pentazocine effect by eliminating a Ca²⁺ trigger arising from activation of L-type VDCC that is necessary for stimulation of release of ER Ca²⁺. A direct linkage between L-type VDCC and sarcoplasmic reticulum Ca²⁺ stores has been demonstrated in muscle tissue (for review, see Putney and Ribeiro, 2000).

Kinases are well established regulators of transporter activity, and several studies have reported linkage between activation of presynaptic receptors and modification of the activity of transporters via a signaling pathway (Apparasundaram et al., 1998; Beckman et al., 1999). Numerous reports have implicated PKC as a signaling mechanism regulating DAT activity. Early studies on cloned transporters showed that phosphorylation of the DAT by phorbol ester activation of PKC decreased the V_max for uptake of [³H]DA by about 20% (Vaughan et al., 1997). Work from the laboratory of Gnegy and colleagues showed that inhibition of PKC with chelerythrine and/or 3-[1-3-(amidonothio)propyl-1H-indol-3-yl]-3-(1-methyl-1H-indol-3-yl)-maleimide blocked amphetamine-stimulated release of endogenous dopamine release and amphetamine-induced motor behavior, but not the uptake of [³H]DA (Browman et al., 1998; Kantor and Gnegy, 1998). In a PC12 model stably transfected with the human DAT, Melikian and Buckley (1999) and Daniels and Amara (1999) showed that phorbol ester activation of PKC resulted in a down-regulation of DAT at the cell surface, attributable to intracellular transporter sequestration. Although these studies show that the DAT contains consensus sequences for recognition by PKC, and that DAT activity can be modified by phosphorylation, the relevant phosphorylation sites that alter DAT expression are not necessarily on the DAT itself. Work on the GAT1 -aminobutyric acid transporter (Corey et al., 1994) and the GLYT1 glycine transporter (Sato et al., 1995), two transporters that, similarly to DAT, contain PKC sequences, showed that when all predicted intracellular PKC sites were mutated, regulation by PKC was left intact, demonstrating that regulation might occur indirectly via other proteins that serve scaffolding or trafficking functions (Sakai et al., 1997). Recently, similar results have been found for the DAT, which also exhibits normal transporter kinetics and trafficking patterns even when all PKC consensus sites on the transporter itself are mutated (Chang et al., 2001). Several target proteins that could be involved in transporter regulation have been suggested, including synapsin 1 and neuromodulin (Iwata et al., 1997), and syntaxin (Beckman et al., 1998). In addition, Kantor et al. (2001) have shown that amphetamine-stimulated DA release via the norepinephrine transporter in PC12 cells is also dependent upon PKC, and is abolished by chelation of calcium. A difference between the findings of Kantor et al. (2000) and our own results is that in our hands, amphetamine-stimulated release itself is not sensitive to PKC inhibition or calcium-dependent. This may be due to the longer incubation time with inhibitors used in the Kantor study compared with ours, or the fact that they measure release of endogenous DA, whereas we measure release of preloaded [³H]DA. Because Kantor et al. (2000) measured release of DA via a norepinephrine transport system, the difference may relate to the transporter studied. In contrast to our findings on control amphetamine-stimulated [³H]DA release, the enhancement by (+)-pentazocine is both abolished by PKC inhibition, and depletion of calcium stores.

Experiments on PC12 cells have shown that this mechanism is governed by the Ca²⁺/CaM kinase II in that model, because inhibitors of this pathway attenuated the (+)-pentazocine enhancement (Weatherspoon and Werling, 1999). Treatment with the Ca²⁺/CaM kinase II inhibitors KN-62 and KN-93 did not change the (+)-pentazocine-mediated enhancement in rat striatal slices, thus indicating that different signaling pathways mediate regulation of DAT activity by ₂-receptors in brain and PC12 cells. It is also possible that these drugs were not able to penetrate slices, although this has not thus far been a problem with other kinase inhibitor tested, nor in other sets of experiments performed in this laboratory, where inhibition of signaling was observed using KN-62 and KN-93 (Weatherspoon and Werling, 1999). Due to negative results obtained by using Ca²⁺/CaM kinase II inhibitors, other signaling pathways were investigated.

Considering the many reports implicating the PKC as a second messenger upon activation of the DAT and other transporters (Kitayama et al., 1994; Qian et al., 1997; Kantor and Gnegy, 1998; Melikian and Buckley, 1999), the role of the PKC was explored as a possible ₂-receptor second messenger. PKC activation is dependent upon Ca²⁺, as is the increase in outward flow of [³H]DA produced by ₂-receptor activation. The treatment of striatal slices with (+)-pentazocine and the selective PKC inhibitor GF109,203X showed a significant decrease to control levels of [³H]DA release. The PKC activator PMA did not produce an additional increase in the (+)-pentazocine-mediated enhancement of amphetamine-stimulated [³H]DA release. PMA alone, however, did produce a significant enhancement of [³H]DA stimulated by amphetamine, an enhancement that was equivalent to that elicited by treatment with (+)-pentazocine alone. This indicates that the same pathway could be activated by both (+)-pentazocine and by PMA, and that the concentrations chosen for each were maximally effective at activation of PKC in this system. Taken together, our results suggest that PKC is a likely participant in the mechanism via which ₂-receptors transduce their signals.

There are several isoforms of PKC, and the inhibitor tested in the current study, GF109203X, is a general inhibitor of the conventional PKCs, which are Ca²⁺-dependent, and novel PKC forms, which are Ca²⁺-independent. The K_i values for inhibition of PKC and PKC are 8.4 and 18 nM, with values at PKC and PKC of 210 and 132 nM, respectively (Way et al., 2000). Studies are currently underway to identify which isozyme of PKC is likely to participate in ₂-receptor-mediated regulation of the DAT. However, because our results with (+)-pentazocine appear to be Ca²⁺-dependent, it is likely that one of the conventional PKCs is involved.

Our study represents one of the first demonstrations of signaling mechanisms activated by ₂-receptor agonists in brain tissue and the first to our knowledge of a -receptor linked via a signaling pathway to transporter regulation. The results are consistent with those of Bowen and colleagues (Vilner and Bowen, 2000), who have demonstrated the association of ₂-receptors with intracellular Ca²⁺ level regulation in neural tumor cells. Our data suggest that changes in intracellular Ca²⁺ levels may ultimately regulate the activity of PKC. The identification of such mechanisms should help elucidate the ultimate role of ₂-receptors in brain functions. We also establish that ₂-receptors can regulate the activity of the DAT. Because of the critical role played by the DAT in the mechanism of action of several drugs of abuse, such as amphetamine and cocaine, it is possible that drugs with antagonist activity at ₂-receptors might be of value in treating drug abuse.