Introduction

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Regulation of catecholamine levels in brain is critical to maintenance of mood and emotional well being. In particular, the reward system is dependent upon dopaminergic activity in the nucleus accumbens. Several drugs of abuse, including amphetamine (AMPH) and cocaine exert their primary effects via actions at the dopamine transporter (DAT), which is responsible for the reaccumulation of ~80% of released dopamine (DA). Recently, much emphasis has been placed on regulation of transporter activity as a means of treating drug abuse.

Receptors are present in dopaminergic brain areas (Gundlach et al., 1986), including striatum and nucleus accumbens. Although the distribution and binding profiles of  receptors are fairly well established, relatively little is known about their function and less about their associated signal transduction mechanisms. We have reported previously that agonists acting at ₂, but not ₁ receptors, enhance [³H]DA release stimulated by AMPH acting via the DAT (Izenwasser et al., 1998). In contrast, agonists acting at  receptors of both the ₁ and ₂ types inhibit release of vesicular [³H]DA from dopaminergic terminal fields in rodent brain (Gonzalez-Alvear and Werling, 1994; Weatherspoon et al., 1996). Measuring the release of catecholamines and the mechanisms underlying the regulation of release in brain tissue presents difficulties including a complex neural network, problems associated with drug penetration, and the presence of multiple types of  receptors. For these reasons, in the present study we have further investigated the interactions between AMPH-stimulated [³H]DA release and  receptors using rat pheochromocytoma (PC12) cells (Greene and Tischler, 1976) as a model system. PC12 cells are of a uniform population and have been reported to bear  receptors primarily of the ₂ type (Hellewell and Bowen, 1990). These investigators did not detect the presence of ₁ receptors in PC12 cells, although a recent report on differentiated PC12 cells discussed the possible expression of the ₁ receptor (Sagi et al., 1996). Undifferentiated PC12 cells are also well characterized in terms of their ability to accumulate, store, and release DA via the DAT (Kadota et al., 1996) and have been used previously to examine second messenger systems involved in vesicular catecholamine release (Shafer and Atchison, 1991). Although a protein with binding characteristics of the ₁ receptor has been cloned (Hanner et al., 1996), at this time no report of ₂ receptor cloning has been made.

In the present study, we show that activation of ₂ receptors by the agonist (+)-pentazocine enhances the ability of AMPH to stimulate [³H]DA release from PC12 cells. The enhancement is reversed by nonsubtype-selective  receptor antagonists and by a ₂ receptor-selective antagonist but not by a ₁ receptor-selective antagonist. We also demonstrate that although the AMPH-stimulated [³H]DA release is not dependent upon exogenous Ca²⁺, addition of the calcium chelator ethylene glycol bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) reduces the enhancement of stimulated release by (+)-pentazocine. We also provide evidence that the enhancement of AMPH-stimulated [³H]DA release by (+)-pentazocine is dependent upon Ca²⁺/calmodulin-dependent protein kinase II (Ca²⁺/CaM kinase II) by showing that two different inhibitors of the enzyme block the enhancing effects of (+)-pentazocine. Together these findings provide the first evidence for a second messenger system associated with  receptor regulation of DAT activity in PC12 cells.

	Materials and Methods

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Chemicals and Reagents. The following drugs and reagents were kindly provided by or obtained from the following sources: AMPH, 1,2-bis(2-amino-phenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester (BAPTA-AM), domperidone, 1,3-di(2-tolyl)guanidine (DTG), 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-[3-phenylpropyl]piperazine dihydrochloride (GBR-12909), 2-[N-(4'-methoxybenzenesulfonyl)]amino-N-(4'-chlorophenyl)-2-propenyl-N-methylbenzylamine phosphate (KN-92), N-[2-[[[3-(4'-chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4'-methoxy-benzene-sulfonamide phosphate (KN-93), nisoxetine hydrochloride, nitrendipine, and yohimbine hydrochloride (Research Biochemicals International, Natick, MA); N-[2-(3,4-dichlorophenyl)-ethyl]N-methyl-2-pyrrolidinyl) ethylamine (BD1008) (Dr. Wayne Bowen and Dr. Brian de Costa, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD); endo-N-(8-methyl-8-azabicyclo[3.2.1.]oct-3-yl)-2,3-dihydro-(1-methyl)ethyl-2-oxo-1H-benzimidazole-1-carboxamidehydrochloride; (BIMU-8) (Dr. Doug Bonhaus, Roche Bioscience, Palo Alto, CA); [³H]DA (specific activity, 46 to 51 Ci/mmol) and [³H]NE (specific activity, 42 Ci/mmol) (Amersham Corp., Arlington Heights, IL); Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (heat-inactivated), penicillin/streptomycin, and trypsin-EDTA (Life Technologies, Inc., Grand Island, NY); 1-(cyclopropylmethyl)-4-2'-4"-fluorophenyl)-2'-oxoethyl)-piperidine hydrogen bromide DuP734 (Drs. William Tam and Rob Zaczek, Du Pont Merck Pharmaceutical Co., Wilmington, DE); EGTA and -conotoxin from Conus geographus (GVIA) (-CgTX; Sigma Chemical Co., St. Louis, MO); horse serum (Biofluids, Rockville, MD); KN-62 (Calbiochem, La Jolla, CA); and (+)-pentazocine (Research Technology Branch, National Institute on Drug Abuse, Rockville, MD).

Tissue Culture of PC12 Cells. Adherent PC12 cells were obtained from Dr. Anne Murphy (Department of Biochemistry, The George Washington University Medical Center, Washington, DC). This line of PC12 cells was originally obtained from John A. Wagner of the Cornell Medical College (New York, NY). The PC12 cells were routinely maintained in 175 cm² culture flasks (Nunclon) in 5% CO₂/95% air at 37°C in DMEM (Life Technologies, Inc.) containing 10% fetal bovine serum (heat-inactivated; Life Technologies, Inc.), 5% horse serum (Biofluids), 100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc.). Cells were fed approximately three times/week and passaged by gentle trituration approximately twice a week at a split ratio of 1:4. Cells were harvested using 0.05% trypsin/0.53 mM EDTA (Life Technologies, Inc.) and counted using 0.04% trypan blue (in PBS) with a Bright-Line hemacytometer (Reichert Scientific Instruments) and an inverted microscope (Cambridge Instruments). Cells of passage number no greater than 20 were used for experiments.

Measurement of [³H]DA Release Using PC12 Cells. Release of [³H]DA from PC12 cells was determined using methods adapted from procedures established in this lab for brain tissue slices (Gonzalez-Alvear and Werling, 1994). PC12 cells were maintained in DMEM and harvested at a density of ~6.0 × 10⁶ to 1.0 × 10⁷ cells/ml for a total cell number of ~1.0 × 10⁸ to 2.0 × 10⁸ cells per experiment, which were ultimately divided into 18 samples. This cell number was found to elicit consistent, measurable DA release in each experiment. The cells were then centrifuged at 1500 rpm for 3 min, and the medium was removed. Cells were resuspended in 20 ml of oxygenated modified Krebs-HEPES buffer containing Mg²⁺ but no Ca²⁺ (MKB: 127 mM NaCl, 5 mM KCl, 1.3 mM NaH₂PO₄, 15 mM HEPES, 10 mM glucose, and 1.2 mM MgSO4; pH adjusted to 7.4 with NaOH). The cell suspension was dispersed using a vortex mixer and again centrifuged at 1500 rpm for 3 min, and the buffer supernatant was removed. The cells were then resuspended in 20 ml ofMKB, gently dispersed using a vortex mixer, and incubated with 15 nM [³H]DA, 0.1 mM ascorbic acid, and 100 nM nisoxetine for 30 min while oxygenated at 37°C. Nisoxetine was included to reduce any contribution of the NE transporter to accumulation of [³H]DA by the PC12 cells. At the end of the 30-min incubation, the cells were centrifuged at 1500 rpm for 3 min, and the supernatant was removed. The cells were resuspended in 20 ml of MKB, centrifuged again, and the supernatant was removed. Cells were then resuspended in a final volume of 5 ml MKB containing 1 µM domperidone (MKD) and gently dispersed by a vortex mixer. Domperidone was included in all subsequent steps of the experiment to prevent activation of DA autoreceptors by the released [³H]DA. The total uptake of [³H]DA was typically between 0.5 and 1 pmol/batch of cells used in an experiment. The cells were then distributed in 250-µl aliquots between glass fiber filter discs into chambers of a BRANDEL (Gaithersburg, MD) superfusion apparatus. MKB was superfused over the tissue at a flow rate of 0.6 ml/min. Buffers were oxygenated throughout the experiments. A low, stable baseline release of ~0.9% per 2-min collection interval was established over a 30-min period. In initial experiments, we established that a 6-min exposure to AMPH as a stimulus was required to produce reproducible, measurable release. After the 6-min S1 period, the inflow was returned to a nonstimulating buffer (interstimulus interval) for a period of 10 min. If a potential inhibitor of release was being tested, it was introduced during this time. The cells were then stimulated a second time for 6 min with AMPH in the presence or absence of another drug as appropriate (stimulus 2; S2). Inflow was again returned to nonstimulating buffer to allow re-establishment of baseline release. The mean fractional [³H]DA release stimulated by 25 µM AMPH in S1 was 3.9 ± 0.28% of total [³H]DA in the tissue at the beginning of the S1. The typical ratio of S2/S1 for AMPH stimulation was 0.6. In experiments to examine for potential effects of EGTA on [³H]DA release, 1 mM EGTA was included in the buffer throughout the experiment. Radioactivity remaining in the tissue was then extracted by a 45-min exposure to 0.2 N HCl. Superfusates were collected at 2-min intervals in scintillation vials, and released radioactivity was determined by liquid scintillation spectroscopy.

	Results

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Our previous results in rat brain tissue revealed that a 10 µM concentration of AMPH produced a consistent, measurable release of preloaded [³H]DA (Izenwasser et al., 1998) from rat striatal slices. We constructed a concentration-response curve for AMPH (0.1-50.0 µM) in PC12 cells and observed a concentration-related increase in stimulation of [³H]DA release, with a fractional release at 25 µM AMPH of 3.9 ± 0.28%. Stimulation with 25 µM AMPH produced a more consistent signal that was enhanced by (+)-pentazocine with a lower variability than stimulation of release by a 10 µM concentration of AMPH, which we had used in brain slices. We therefore adopted 25 µM AMPH as our standard stimulus.

We have determined previously that, at the concentrations that produced enhancement of AMPH-stimulated [³H]DA release from slices of rat caudate putamen, (+)-pentazocine (10 nM to 100 µM) had no effect on the uptake of [³H]DA in either slices or synaptosomes prepared from the same brain region (Izenwasser et al., 1998). In the present study, we tested a range of concentrations of (+)-pentazocine on [³H]DA release stimulated by 25 µM AMPH in PC12 cells. In these experiments, the higher concentrations of (+)-pentazocine (500 to 1000 nM) significantly enhanced release stimulated by AMPH (Fig. 1), suggesting that activation of ₂ receptors was more likely responsible for the enhancement than activation of ₁ receptors.

View larger version (70K): [in this window] [in a new window]   Fig. 1.   Effects of (+)-pentazocine on AMPH-stimulated [3H]DA release from PC12 cells. Data are expressed as percentage of control-stimulated release with baseline release subtracted. Release of [3H]DA was stimulated by 25 µM AMPH in the presence of the indicated concentration of (+)-pentazocine. *Significantly different from control by ANOVA with posthoc Dunnett's (p < .05). Statistical analysis was performed on untransformed data (S2/S1) as described in Materials and Methods; n = 7.

In additional experiments, we sought to confirm whether the enhancement by (+)-pentazocine was mediated via activation of either ₁ or ₂ receptors. In brain tissue, (+)-pentazocine has a K_i at ₁ receptors of 2-6 nM and at ₂ receptors of about 1.5 µM (Vilner and Bowen, 1992). In PC12 cells, the K_i for (+)-pentazocine at ₂ receptors has been reported to be 1 µM (Hellewell and Bowen, 1990). We tested the effects of several  receptor antagonists at concentrations chosen to occupy 50% of their preferred  receptor subtype on [³H]DA release stimulated by 25 µM AMPH in the presence of 500 nM (+)-pentazocine. The ₂ receptor antagonists BIMU-8 (100 nM), as well as the nonsubtype selective  antagonists DTG (100 nM) and BD1008 (10 nM), each produced a significant reversal of the enhancement of AMPH-stimulated release by 500 nM (+)-pentazocine (Fig. 2). In contrast, the ₁ receptor-selective antagonist DuP734 (100 nM) did not have a significant effect on the enhancement of release by (+)-pentazocine. No  receptor antagonist tested had any significant effect on basal release. Basal fractional release in the absence of drug was 0.9 ± 0.21% per 2-min collection interval, whereas basal fractional release for the same time interval in the presence of BIMU-8 was 0.84 ± 0.42%, in the presence of DuP734 was 1.3 ± 0.84%, in the presence of DTG was 1.0 ± 0.47%, and in the presence of BD1008 was 1.8 ± 0.9%.

View larger version (52K): [in this window] [in a new window]   Fig. 2.   Effects of  receptor antagonists on (+)-pentazocine-mediated enhancement of 25 µM AMPH-stimulated [3H]DA release from PC12 cells. Release was stimulated by 25 µM AMPH in the presence or absence of (+)-pentazocine (500 nM) and a  receptor antagonist as indicated. Concentrations of drugs were: BIMU-8, 100 nM; DuP734, 100 nM; DTG, 100 nM; and BD1008, 10 nM. Data are expressed as percentage of release stimulated by AMPH alone (percentage of control-stimulated release). *Enhancement of stimulated release by (+)-pentazocine (500 nM) alone was significantly different from control by ANOVA and posthoc Dunnett's (two-tailed; p < .05). #Enhancement by (+)-pentazocine alone was significantly different from release in the presence of (+)-pentazocine plus antagonist by ANOVA and posthoc Dunnett's (two-tailed; p < .05). Statistical analysis was performed on untransformed data (S2/S1) as described in Materials and Methods; n = 3.

Because DA is the precursor for the synthesis of NE, we also tested whether activation of ₂ receptors could regulate the release of [³H]NE via the norepinephrine transporter (NET). We observed that the total uptake of [³H]NE was low in these experiments, as compared with the total uptake of [³H]DA observed in other experiments using PC12 cells; the total uptake (mean ± S.D.) of [³H]NE was 18.3 ± 4.9% of total [³H]DA uptake under the same conditions (n = 2). AMPH did not consistently stimulate a reliably measurable amount of [³H]NE release, probably due to the low accumulation of transmitter. We did not detect any effect of (+)-pentazocine on [³H]NE release stimulated by 25 µM AMPH; the mean values ± S.D. for percentage of stimulated release were 100 ± 10.8% (AMPH alone), 91.6 ± 16.1% [AMPH + 500 nM (+)-pentazocine], and 95.6 ± 7.3% [AMPH + 1 µM (+)-pentazocine] (n = 2). These results suggest that the (+)-pentazocine-mediated enhancement of AMPH-stimulated [³H]DA release we observed in PC12 cells was due to actions at the DAT and not the NET.

Because we have shown previously that chelation of Ca²⁺ with EGTA prevents the (+)-pentazocine-mediated enhancement of AMPH-stimulated [³H]DA release from rat striatal slices by  receptor agonists (Izenwasser et al., 1998), we performed experiments to determine whether Ca²⁺ would similarly be required for the (+)-pentazocine-mediated effect in PC12 cells. In this set of experiments, (+)-pentazocine again produced an enhancement of AMPH-stimulated [³H]DA release at the higher concentrations tested (500 to 1000 nM) as compared with control (p < .05 by ANOVA with posthoc Dunnett's), as found previously. In the presence of 1 mM EGTA, (+)-pentazocine did not produce a significant enhancement of [³H]DA release stimulated by 25 µM AMPH at any concentration tested (Fig. 3). A significant difference was detected comparing the effect of 500 nM and 1000 nM (+)-pentazocine in the presence versus the absence of EGTA. These results suggest that Ca²⁺ is required for (+)-pentazocine to enhance release stimulated by AMPH in PC12 cells. We also tested the intracellular Ca²⁺ chelator BAPTA-AM (10 µM) in an attempt to identify the source of Ca²⁺ required to support the (+)-pentazocine-mediated effect. Although the BAPTA-AM treatment did not affect cell viability as measured by trypan blue exclusion, the inclusion of BAPTA-AM in the superfusion buffer greatly reduced the ability of AMPH to stimulate release, making it impossible to detect whether it specifically influenced the effect of (+)-pentazocine on release. We therefore chose other approaches to investigate the potential role of calcium in the (+)-pentazocine-mediated effects on AMPH-stimulated [³H]DA release.

View larger version (39K): [in this window] [in a new window]   Fig. 3.   Effects of EGTA in the presence or absence of (+)-pentazocine on AMPH-stimulated [3H]DA release from PC12 cells. Data are expressed as percentage of control-stimulated release with baseline release subtracted. Release of [3H]DA was stimulated by 25 µM AMPH in the presence or absence of (+)-pentazocine and in the absence (hatched columns) or presence (open columns) of EGTA (1 mM). *Significantly different from control by ANOVA with posthoc Dunnett's (p < .05) and also different from the same concentrations of (+)-pentazocine in the presence of EGTA. There was no significant effect of (+)-pentazocine compared with control at any concentration in the presence of EGTA. Statistical analysis was performed on untransformed data (S2/S1) (see Materials and Methods); n = 7 for control and 4 for EGTA.

If the Ca²⁺ required for the (+)-pentazocine-mediated effect in PC12 cells originates externally, it might enter via a voltage-dependent calcium channel (VDCC). Both L- and N-type VDCC are present in PC12 cells (Shafer and Atchison, 1991). We evaluated the effect of nitrendipine, a selective inhibitor of L-type VDCC, as well as the selective N-type VDCC blocker -conotoxin GVIA (-CgTX) on  receptor regulation of AMPH-stimulated [³H]DA release. Both compounds were tested at 100 nM, a concentration that should produce complete blockade of the respective VDCC. Whereas nitrendipine alone had no effect compared with control-stimulated release, -CgTX produced a slight enhancement of release in the absence of (+)-pentazocine, but this effect was not statistically significant. As seen previously, (+)-pentazocine (1 µM) alone significantly enhanced [³H]DA release stimulated by 25 µM AMPH, compared with release stimulated by AMPH alone (Fig. 4). The release in the presence of (+)-pentazocine + -conotoxin was also significantly different from control stimulated release. Neither nitrendipine nor -CgTX had any significant effect on the enhancement mediated by (+)-pentazocine. Thus, it appeared that neither the L- nor the N- type VDCC was responsible for supplying the Ca ²⁺ needed to support the enhancing effect of (+)-pentazocine on AMPH-stimulated release.

View larger version (52K): [in this window] [in a new window]   Fig. 4.   Effects of nitrendipine (NTP) and -CgTX on (+)-pentazocine-mediated enhancement of AMPH-stimulated [3H]DA release from PC12 cells. Release was stimulated by 25 µM AMPH in the presence or absence of (+)-pentazocine (1 µM) and nitrendipine (100 nM) or -CgTX (100 nM) as indicated. Data are expressed as a percentage of release stimulated by AMPH alone (percentage of control-stimulated release). *Significantly different from control by ANOVA and posthoc Dunnett's at p < .05. Statistical analysis was performed on untransformed data (S2/S1) as described in Materials and Method; n = 5 for control and (+)-pentazocine treatments, n = 3 for other treatments. There were no significant differences detected among (+)-pentazocine alone and (+)-pentazocine in the presence of either VDCC blocker.

We also tested inhibitors of Ca²⁺/CaM kinase II for potential effects on (+)-pentazocine-mediated enhancement of AMPH-stimulated [³H]DA release. In experiments to examine the effects of KN-62, (+)-pentazocine (1 µM) alone produced a significant enhancement of [³H]DA release stimulated by 25 µM AMPH, compared with release stimulated by AMPH alone (p < .05; two-way ANOVA and posthoc Dunnett's test) (Fig. 5). Enhancement of AMPH-stimulated release by 1 µM (+)-pentazocine was significantly attenuated in the presence of 10 µM KN-62. This same concentration of KN-62 alone had no effect on control AMPH-stimulated release. The vehicle in which KN-62 was dissolved, DMSO, had no effect on either unstimulated or AMPH-stimulated release.

View larger version (58K): [in this window] [in a new window]   Fig. 5.   Effects of KN-62 on (+)-pentazocine-mediated enhancement of AMPH-stimulated [3H]DA release from PC12 cells. Release was stimulated by 25 µM AMPH in the presence or absence of (+)-pentazocine (1 µM) and KN-62 (10 µM) or DMSO (the vehicle for KN-62) as indicated. Data are expressed as percentage of release stimulated by AMPH alone (percentage of control-stimulated release). *Enhancement of stimulated release by (+)-pentazocine (1 µM) alone was significantly different from control by ANOVA and posthoc Dunnett's (two-tailed; p < .05). #Enhancement by (+)-pentazocine alone was significantly different from release in the presence of (+)-pentazocine plus KN-62 by ANOVA and posthoc Dunnett's (two-tailed; p < .05) Statistical analysis was performed on untransformed data (S2/S1) as described in Materials and Methods; n = 3.

We also evaluated the effects of another selective inhibitor of Ca²⁺/CaM kinase II, KN-93, a compound that is structurally dissimilar from KN-62 (Sumi et al., 1991), as well as KN-92, a structural analog of KN-93 that does not inhibit Ca²⁺/CaM kinase II and has been used as a negative control for KN-93 (Pierce and Kalivas, 1997). We chose a concentration of 10 µM for both KN-93 and KN-92, based on a reported inhibition constant (K_i) obtained from binding studies (K_i = 0.37 µM; Sumi et al., 1991). At 10 µM, neither compound tested alone had any effect on control-stimulated release (Fig. 6). The enhancement of AMPH-stimulated release mediated by 1 µM (+)-pentazocine was significantly attenuated by the presence of a 10 µM concentration of KN-93. In contrast, 10 µM KN-92 had no effect on the enhancement of AMPH-stimulated release mediated by (+)-pentazocine.

View larger version (63K): [in this window] [in a new window]   Fig. 6.   Effects of KN-92 and KN-93 on (+)-pentazocine-mediated enhancement of AMPH-stimulated [3H]DA release from PC12 cells. Release was stimulated by 25 µM AMPH in the presence or absence of (+)-pentazocine (1 µM) and KN-92 (10 µM) or KN-93 (10 µM) as indicated. Data are expressed as percentage of release stimulated by AMPH alone (percentage of control-stimulated release). *Enhancement of stimulated release by (+)-pentazocine (1 µM) alone was significantly different from control by ANOVA and posthoc Dunnett's (two-tailed; p < .05). #Enhancement by (+)-pentazocine alone was significantly different from release in the presence of (+)-pentazocine plus KN-93 by ANOVA and posthoc Dunnett's (two-tailed; p < .05) Statistical analysis was performed on untransformed data (S2/S1) as described in Materials and Methods; n = 3.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Previous studies support the role of the DAT in regulation of catecholamine function in brain (Giros and Caron, 1993). The DAT is a target for drugs of abuse; therefore, regulation of the DAT could be therapeutically beneficial. Because we had found that in rat striatal tissue, (+)-pentazocine, acting via the pharmacologically identified ₂ receptor, enhanced outward transport of DA via the DAT (Izenwasser et al., 1998), we modeled the apparent regulation in PC12 cells, where study of underlying mechanisms might be facilitated.

Undifferentiated PC12 cells synthesize and release DA and NE (Koike and Takashima, 1986) and bear both the DAT and the NET (Shafer and Atchison, 1991; Kadota et al., 1996). Hellewell and Bowen (1990) identified receptors with a ₂-like pharmacology in PC12 cells. We now report that the agonist (+)-pentazocine, acting at ₂ receptors, can modulate AMPH-stimulated release of [³H]DA from PC12 cells; we believe this system to be a novel model for ₂ receptor function.

We used AMPH, a substrate for the DAT, to stimulate the release of [³H]DA. The stimulant effects of AMPH are caused by the release of biogenic amines from central nervous system nerve terminals (Philips et al., 1982; Sulzer et al., 1995). A two-component model of AMPH-stimulated release postulates that AMPH stimulates DA release from synaptic vesicles to the cytosol, which results in reverse transport and release of DA via the plasma membrane DAT (Schuldiner et al., 1993; Pifl et al., 1995; Sulzer et al., 1995; Floor and Meng, 1996).

We demonstrated previously that (+)-pentazocine at concentrations >100 nM enhances AMPH-stimulated [³H]DA release from slices of rat caudate putamen (Izenwasser et al., 1998). Additionally, we demonstrated that concentrations of (+)-pentazocine between 10 nM and 10 µM produced no effect on [³H]DA uptake. Also, (+)-pentazocine (10 nM to 10 µM) had no effect on basal DA release. If (+)-pentazocine were inhibiting reuptake, it should also enhance apparent basal DA release. In the present study, concentrations of (+)-pentazocine 500 nM significantly enhanced AMPH-stimulated [³H]DA release from PC12 cells.

The enhancing effects of (+)-pentazocine on AMPH-stimulated [³H]DA release appear to be specific to the DAT. Nisoxetine was always present during the incubation period with [³H]DA to prevent uptake via the NET. Also, in experiments to assess potential contribution of conversion of [³H]DA to [³H]NE, there was little uptake of [³H]NE, and no measurable effect of (+)-pentazocine on AMPH-stimulated [³H]NE release under the conditions used in this study.

At concentrations of (+)-pentazocine that enhanced AMPH-stimulated [³H]DA release, ₂ receptors would be activated, because the K_i of (+)-pentazocine at ₂ has been reported as 1.5 µM in rat brain tissue (Vilner and Bowen, 1992), 1.3 µM in rat liver (Hellewell et al., 1994), and 1 µM in PC12 cells (Hellewell and Bowen, 1990). A higher concentration of (+)-pentazocine was required to enhance AMPH-stimulated release in PC12 cells than we found using rat striatum, although the reasons for this difference are unclear. It is possible that the receptors classified as ₂ in the various tissues are not absolutely identical, despite their very similar pharmacological sensitivities. In neither brain nor PC12 cells did concentrations of (+)-pentazocine that would preferentially activate ₁ receptors produce any effect on AMPH-stimulated or basal [³H]DA release. The ₁ receptor has a K_i for (+)-pentazocine of between 2 and 6 nM (Walker et al., 1990); therefore, it is unlikely that ₁ receptors contributed to the (+)-pentazocine-mediated enhancement. The ability of a 100 nM concentration of the ₂-selective antagonist BIMU-8 (Weatherspoon et al., 1997) at a concentration that should occupy 80% of ₂ receptors (K_i at ₂ = 20 nM), but no ₁ receptors (K_i = 6.9 µM; Bonhaus et al., 1993), to reverse the (+)-pentazocine-mediated enhancement supports the identification of the receptor involved as ₂. Likewise, the nonsubtype-selective  receptor antagonists DTG at 100 nM (K_i at ₂ = 38 nM; Walker et al., 1990) and BD1008 at 10 nM (K_i at  undifferentiated for subtype = 1.2 nM) both fully reversed the (+)-pentazocine-mediated enhancement. The reversal of the effect of (+)-pentazocine by DTG in the present study is in contrast to our finding of a slight but nonsignificant reversal by DTG of the (+)-pentazocine effect in our previous study (Izenwasser et al., 1998). Perhaps in our earlier study, increasing the concentration of DTG would have produced significant reversal. The reasons for the discrepancy are unclear but may be related to slight differences in the sensitivity of ₂ receptors in PC12 cells. It also bears noting that although DTG behaves as a  antagonist in our own assays of neurotransmitter release, others have identified -agonist activity of DTG. In contrast to the actions of ₂ antagonists in the present study, the ₁-selective antagonist DuP734 (K_i at ₁ = 10 nM, K_i at ₂ >1 µM; Culp et al., 1992) did not significantly reduce the (+)-pentazocine-mediated enhancement of stimulated release. It should be noted that whereas Ca²⁺ has long been known to be required for vesicular transmitter release, the potential role of Ca²⁺ in transporter-mediated transmitter release remains to be determined (Bowyer et al., 1984). Several studies indicate that transporter-dependent stimulation of DA release in response to AMPH may occur in the absence of exogenous Ca²⁺, although release of Ca²⁺ from internal stores may be critical (Sulzer et al., 1995; Wall et al., 1995).

Ca²⁺ may be important for receptor-mediated regulation of DA uptake and release via the DAT. Ca²⁺ plays a role in the effects of nicotine, mediated via nicotinic acetylcholine receptors, on DA uptake in PC12 cells that possess the DAT (Yamashita et al., 1995), suggesting that ligands acting via other receptor types might also affect transporter-mediated uptake and release in a Ca²⁺-dependent manner. Furthermore, the DAT is known to possess a number of phosphorylation sites that may be phosphorylated by Ca²⁺-dependent intracellular second messenger molecules such as protein kinase C, thus affecting DAT function (Giros and Caron, 1993).

The reduction in enhancement of (+)-pentazocine-mediated enhancement of AMPH-stimulated [³H]DA by 1 mM EGTA release in both striatal tissue (Izenwasser et al., 1998) and PC12 cells suggested that Ca²⁺ was required for that effect. These results do not indicate whether the source(s) of Ca²⁺ important for the enhancement is extracellular and/or intracellular, because chelating extracellular Ca²⁺ might result in secondary changes in intracellular Ca²⁺ levels. Attempts to elucidate the role of intracellular Ca²⁺ using BAPTA-AM were unsuccessful; therefore, we tested whether compounds that interfere with L- and N-type VDCC would affect ₂ receptor-mediated regulation of AMPH-stimulated DA release. Neither nitrendipine nor -CgTX had a significant effect, although -CgTX itself slightly, but not significantly, enhanced release above basal. Therefore, entry of Ca²⁺ through L- or N-type VDCC did not appear to be the source of Ca²⁺ for the (+)-pentazocine-mediated effect.

Previous studies have demonstrated that depletion of intracellular stores of Ca²⁺, in response to receptor-mediated activation of the IP₃ second messenger pathway and subsequent IP₃-mediated release of [Ca²⁺]_i, activates Ca²⁺ entry across the plasma membrane. This has been referred to as capacitative Ca²⁺ entry (Clapham, 1995; Ko et al., 1996). Capacitative Ca²⁺ entry increases cytoplasmic Ca²⁺ in response to depletion of intracellular Ca²⁺ stores and has potentially important roles in many Ca²⁺-dependent cellular functions (Ghosh and Greenberg, 1995). In support of this hypothesis that EGTA may affect capacitative Ca²⁺ entry in our study, thapsigargin, a Ca²⁺-ATPase inhibitor, has been shown to mediate an EGTA-sensitive increase in [Ca²⁺]_i via capacitative Ca²⁺ entry in other cell models (Ko et al., 1996; Louzao et al., 1996). Chelation of extracellular Ca²⁺ with EGTA has also been associated with Ca²⁺ extrusion from rat parotid acinar cells, resulting in decreased [Ca²⁺]_i (Takemura et al., 1990). Ligands acting at ₂ receptors can release Ca²⁺ from intracellular stores in indo-1-loaded human SK-N-SH neuroblastoma cells (Vilner and Bowen, 1995; Bowen et al., 1996). If (+)-pentazocine mediates Ca²⁺ release from intracellular stores, this might activate capacitative Ca²⁺ entry across the plasma membrane. In that case, the enhancement of AMPH-stimulated DA release by (+)-pentazocine would require both the release of [Ca²⁺]_i and subsequent capacitative Ca²⁺ entry. EGTA, via chelation of extracellular Ca²⁺, would remove the Ca²⁺ necessary for capacitative Ca²⁺ entry across the plasma membrane of the PC12 cells, making the net effect of EGTA a decrease in the cytoplasmic Ca²⁺ available for (+)-pentazocine to mediate enhancement of AMPH-stimulated DA release.

EGTA has also been shown to modify physiological effects produced by AMPH. Administration of EGTA to mice has been shown to inhibit AMPH-induced circling behavior, which indicates a role for Ca²⁺ in the response (Fung and Schwarz, 1983). These studies did not assess the relative contributions of extracellular and/or intracellular Ca²⁺ in the effects mediated by AMPH.

In conclusion, we report a model of ₂ receptor function using PC12 cells. This represents the first such model that enables identification of signal transduction mechanisms involved in ₂ receptor regulation of catecholamine release. Our results suggest that (+)-pentazocine, acting via a ₂ receptor, enhances AMPH-stimulated DA release either by directly altering the function of the DAT or by mobilizing a vesicular pool of DA by altering the outward flux of DA through the vesicular amine transporter. We are currently investigating these possibilities. Our results suggest an important role for Ca²⁺/CaM kinase II in ₂ receptor regulation of AMPH-stimulated DA release. ₂ receptors may be important in the effects of AMPH that are associated with drug abuse and other conditions that involve enhanced levels of synaptic DA. For instance, recent evidence from positron emission tomography scans suggests that schizophrenia is associated with enhanced AMPH-induced DA release in both drug naive and antipsychotic-treated patients (Breier et al., 1997).