ς2-Receptor Regulation of Dopamine Transporter via Activation of Protein Kinase C

  1. Alicia E. Derbez,
  2. Rupal M. Mody and
  3. Linda L. Werling
  1. Department of Pharmacology, The George Washington University Medical Center, Washington, DC
  1. Dr. Linda L. Werling, Department of Pharmacology, The George Washington University Medical Center, 2300 Eye St. NW, Washington, DC 20037. E-mail: phmllw{at}gwumc.edu
 
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Abstract

The elucidation of the mechanisms underlying ς2-receptor activation and signal transduction is crucial to the understanding of ς2-receptor function. Previous studies in our laboratory have demonstrated ς2-receptor-mediated regulation of the dopamine transporter (DAT) as measured by amphetamine-stimulated release of [3H]dopamine (DA) from both rat striatal slices and PC12 cells. The regulation of the DAT in the PC12 cell model was dependent upon activation of Ca2+/calmodulin-dependent kinase II. We have now studied the second messenger systems involved in ς2-receptor-mediated regulation of amphetamine-stimulated [3H]DA release in rat striatal slices, including Ca2+/calmodulin-dependent kinase II, protein kinase C, and sources of calcium required for the enhancement of release produced by ς2-receptor activation. The Ca2+/calmodulin-dependent kinase II inhibitors 1-[N,O-bis-(5-isoquionolinesulfonyl)]-N-methyl-l-tyrosyl-4-phenylpiperazine andN-[2-[[[3-(4′-chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4′-methoxy-benzenesulfonamide phosphate did not significantly affect the (+)-pentazocine-mediated enhancement of amphetamine-stimulated [3H]DA release. However, we found that an inhibitor of protein kinase C, 3-[1-[3-(dimethylamino)propyl]-1H-indol-3-yl)-1H-pyrrole-2,5-dione, blocks the (+)-pentazocine-mediated enhancement in rat striatal slices. The protein kinase C activator phorbol 12-myristate 13-acetate, but not the inactive isophorbol 4α,9α,12α,13α,20-pentahydroxytiglia-1,6-dien-3-one, enhanced the amphetamine-stimulated [3H]DA release comparable to the enhancement seen by (+)-pentazocine alone. Additionally, the L-type voltage-dependent calcium channel inhibitor nitrendipine or prior treatment with thapsigargin, but not the N-type voltage-dependent calcium channel ω-conotoxin MVIIA, attenuated the (+)-pentazocine-mediated enhancement. Together, these data suggest that activation of ς2-receptors results in the regulation of DAT activity via a calcium- and protein kinase C-dependent signaling mechanism.

ς-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 ς-receptors, designated ς1 and ς2 (Hellewell and Bowen, 1990; Quirion et al., 1992). ς-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. ς1- and ς2-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). ς1-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).

ς1-Receptors have been associated with second messenger activation and inhibition in several model systems. There is disagreement whether ς1-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 ς1-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. ς1-Receptors have been associated with calcium homeostasis (Brent et al., 1997; Klette et al., 1997; Hayashi et al., 2000).

To date, the ς2-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 ς2-receptors, and a reversal of that increase in intracellular calcium by two ς-receptor antagonists. ς2-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, seeBowen, 2000).

In this laboratory, we have studied modulation of catecholamine release by ς receptor ligands acting through ς1- and ς2-receptors (Gonzalez-Alvear and Werling, 1994, 1995; Izenwasser et al., 1998; Weatherspoon and Werling, 1999). The ς-selective agonist (+)-pentazocine has aKi for binding to ς2-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 ς2-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 ς2-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 ς2-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 ς2-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.

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Materials and Methods

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 (Table1). 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).

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Table 1

Lack of effect of (+)-pentazocine on uptake of [3H]dopamine into rat striatal synaptosomes

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.

In our initial studies designed to investigate the effects of drugs that interfered with signaling mechanisms that might be associated with ς-receptors in rat striatal slices, we found the variability around measurement of amphetamine-stimulated [3H]DA release to be sometimes greater than 10% around the mean. We hypothesized that this variability could be due to interference from interneurons, and therefore added tetrodotoxin (TTX) at a concentration of 1 μM to the buffer. TTX blocks sodium channels, thereby preventing contributions to the measured responses by action potentials propagated through interneurons. The addition of TTX decreased the variability to less than 10%.

MKB containing 1 μM TTX was perfused over the tissue at a flow rate of 0.6 ml/min. A low, stable baseline release of approximately 1.1%/min was established over a 30-min period. The tissue was then stimulated to release [3H]DA by a 2-min exposure to 10 μM AMPH (stimulus 1, S1). The inflow was then returned to a nonstimulating buffer (interstimulus interval, ISI) for a period of 10 min. If the effect of a drug on stimulated release was being examined (such as ς-receptor antagonists, VDCC blockers, or Ca2+/CaM kinase II inhibitors), the drug was introduced at this time. For the GF109,203X time course study, 100 nM GF109,203X was introduced at the times indicated. The tissue was then stimulated a second time for 2 min with AMPH in the presence or the absence of drug being tested, as appropriate (stimulus 2, S2). Inflow was then returned to nonstimulating buffer to allow a return to baseline release before extraction of the radioactivity remaining in the tissue through a 45-min exposure to 0.2 N HCl. Under our experimental conditions, we have previously shown that amphetamine-stimulated release of [3H]DA is completely blocked by nomifensine (Drew and Werling, 2001).

Superfusates were collected at 2-min intervals in scintillation vials, and the glass fiber filter discs and tissue were collected into the final vials. Released radioactivity was determined by liquid scintillation spectroscopy. Data are expressed as radioactivity released above baseline during the collection interval as a percentage of the radioactivity released by control stimulus (%control-stimulated release). Control-stimulated release was 7.98 ± 0.09% S.E.M. (S.D. = 0.62%; coefficient of variation = 7.78%;N = 38). In experiments on the effects on the enhancement or inhibition of drugs, data were statistically analyzed as ratios (S2/S1; typically 0.8 for AMPH alone and 1.2 when (+)-pentazocine is included in S2) before transformation of data into percentage of control-stimulated release for facilitating comparison across treatments. Drugs include ς-receptor agonists and antagonists, and drugs that modulate Ca2+ signaling. Data were analyzed using one-way factorial ANOVA with post hoc Dunnett's tests. Results were considered to be significantly different whenp < 0.05.

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, ω-conotoxin, thapsigargin, and 4α-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); andendo-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).

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Results

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 and2). 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 Ki for (+)-pentazocine binding to ς2-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 theKi for (+)-pentazocine at the ς2-receptor. This should produce an occupancy of 67% if the Ki is closer to the 500 nM value (Quirion et al., 1992) and 47% if theKi value is 1.5 μM (Vilner and Bowen, 1992) of ς2-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 ς2-selective antagonist BIMU-8 (Ki for ς2, 20 nM; Ki for ς1, >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).

Figure 1
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Figure 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 att = 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.

Figure 2
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Figure 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 ς2-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 ς2-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 (Ki 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 ς2 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 (Ki = 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%)

Figure 3
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Figure 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 (Ki 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 ω-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 ω-conotoxin produced 130 ± 20% (N = 3).

Figure 4
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Figure 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.

We also tested whether prior depletion of ER Ca2+stores by thapsigargin would affect the ability of (+)-pentazocine to enhance amphetamine-stimulated [3H]DA release from striatal slices. Thapsigargin pretreatment (2 μM during the final 5-min wash after incubation with [3H]DA) completely abolished in the ability of (+)-pentazocine to enhance amphetamine-stimulated release (Fig. 4B).

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 ς2-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.

Figure 5
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Figure 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.

We also tested the time course for inhibition of the (+)-pentazocine-mediated enhancement by GF109203X (Fig. 6). GF109,203X was introduced at one of the following time points: concomitantly with introduction of the first stimulus by amphetamine (S1), during the ISI, concomitantly with the introduction of (+)-pentazocine, or concomitantly with the second amphetamine stimulus (S2). The results show that even inhibition of PKC simultaneously with application of the second amphetamine stimulus is sufficient to block enhancement of amphetamine-stimulated [3H]DA release.

Figure 6
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Figure 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.

Direct stimulation of PKC with 10 μM PMA alone (Fig. 7) showed an enhancement of the amphetamine-stimulated [3H]DA release comparable to the enhancement by (+)-pentazocine alone, consistent with a role for PKC in the regulation of outward activation of the DAT. The combination of 1 μM (+)-pentazocine with 10 μM PMA did not produce greater stimulation than either (+)-pentazocine or PMA alone. In contrast, the inactive analog of PMA, 4α-phorbol at 10 μM had no effect on amphetamine-stimulated [3H]DA release.

Figure 7
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Figure 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 4α-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.

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Discussion

The elucidation of the mechanisms underlying ς2-receptor activation and signal transduction are crucial to the understanding of ς2-receptor function. The goal of this study was to identify the signaling pathway(s) through which ς2-receptors regulate the outflow of [3H]DA via the DAT in slices of rat striatum.

Our data show that the activation of ς2-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 ofIzenwasser et al. (1993).

To our knowledge, the only previously identified signaling system associated with ς2-receptors has been regulation of intracellular Ca2+levels as demonstrated by Vilner and Bowen (2000). They demonstrated that ς2-ligands produced an immediate, dose-dependent, and transient rise in intracellular Ca2+ originating from the ER that was blocked by the ς-receptor antagonistsN-[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 ς2-agonists that was thapsigargin-insensitive.

The enhancement of amphetamine stimulated [3H]DA release via activation of ς2-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 ς2-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 ς2-receptor activation. The L-type VDCC inhibitor nitrendipine, but not the N-type inhibitor ω-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 ς2-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 Vmax 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 γ-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 ς2-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 ς2-receptor second messenger. PKC activation is dependent upon Ca2+, as is the increase in outward flow of [3H]DA produced by ς2-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 ς2-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. TheKi values for inhibition of PKCα and PKCβ are 8.4 and 18 nM, with values at PKCδ and PKCε 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 ς2-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 ς2-receptor agonists in brain tissue and the first to our knowledge of a ς-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 ς2-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 ς2-receptors in brain functions. We also establish that ς2-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 ς2-receptors might be of value in treating drug abuse.

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Acknowledgments

We thank Dr. Doug Bonhaus for the gift of BIMU-8. We thank Dr. John K. Weatherspoon for performing some of the experiments. We also appreciate the critical review of the manuscript by Dr. David Perry.

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Footnotes

  • This study was supported by National Institute on Drug Abuse Grant DA 06667 (to L.L.W.) and National Institute on Drug Abuse Predoctoral Fellowship DA 06002 (to A.E.D.). A.E.D. is a predoctoral student in the Department of Pharmacology, The George Washington Institute for Biomedical Sciences. This work will be part of a dissertation presented to the above-mentioned department in partial fulfillment of the requirements for the Ph.D. degree. The findings herein were reported in preliminary form in Derbez AE, Mody RM, Weatherspoon JK, and Werling LL (1999) Mechanisms by which sigma2 (ς2) receptors may regulate dopamine release ([3H]DA release) from slices of brain tissue and cells in culture. Soc Neurosci Abstr25:1475 and Derbez AE and Werling LL (2000) Regulation of dopamine transporter via sigma-2 receptor activation of calcium-dependent second messengers.FASEB Abstr14:1372.

  • Abbreviations:
    SKF10,047
    N-allylnormetazocine
    ER
    endoplasmic reticulum
    AMPH
    amphetamine
    DA
    dopamine
    Ca2+/CaM kinase II
    Ca2+/calmodulin-dependent protein kinase II
    DAT
    dopamine transporter
    PKC
    protein kinase C
    SUP
    supernatant
    MKB
    modified Krebs-HEPES buffer
    ANOVA
    analysis of variance
    TTX
    tetrodotoxin
    S1
    first ampthetamine stimulus
    ISI
    interstimulus interval
    S2
    second amphetamine stimulus
    VDCC
    voltage-dependent calcium channel
    GF109,203X
    3-[1-[3-(dimethylamino)propyl]-1H-indol-3-yl)-1H-pyrrole-2,5-dione
    KN-62
    1-[N,O-bis-(5-isoquionolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine
    KN-92
    2-[N-(4′-methoxybenzenesulfonyl)]amino-N-(4′chlorophenyl)-2-propenyl-N-methylbenzylamine phosphate
    KN-93
    N-[2-[[[3-(4′-chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4′-methoxy-benzenesulfonamide phosphate
    PMA
    phorbol-12-myristate-13-acetate
    NIT
    nitrendipine
    BIMU-8
    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
    4α-phorbol
    isophorbol (4α,9α,12α,13α,20-pentahydroxytiglia-1,6-dien-3-one)
    • Received August 24, 2001.
    • Accepted December 13, 2001.
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