Introduction

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Receptors are widely distributed in brain and peripheral tissues and have been localized to plasma membrane (PM) as well as intracellular structures, including endoplasmic reticulum (ER) (McCann et al., 1994; Alonso et al., 2000).  Receptors have been linked with a variety of physiological functions, including neuroprotection (for review, see Maurice et al., 1999), analgesia (Chien and Pasternak, 1994), memory (Maurice and Lockhart, 1997; Urani et al., 1998), gastric acid balance (Hara et al., 1994), and actions of antipsychotic drugs (Romero et al., 2000). Little has been reported regarding the linkage of  receptors to specific signaling pathways. Bowen et al. (1988) first reported that  receptor ligands inhibited muscarinic-stimulated phosphoinositide turnover. Vilner and Bowen (2000) later showed that ligands at ₂ receptors regulated ER Ca²⁺ homeostasis in NG108 cells, and Su and coworkers have demonstrated a link between ₁ receptors and ER Ca²⁺ via interaction with the cytoskeletal protein ankyrin in SK-N-SH cells (Hayashi et al., 2000; Hayashi and Su, 2001). We have also shown that  receptor activation similarly regulates ER Ca²⁺ homeostasis in SH-SY5Y cells (Hong, 2002). Finally, Novakova et al. (1998) reported a stimulation of IP₃ production by  ligands in rat cardiac myocytes. Collectively, these findings suggest that  receptor signaling could occur via the PLC/PKC system.

Our previous data (Gonzalez-Alvear and Werling, 1994, 1995; Weatherspoon et al., 1996; Ault and Werling, 1997, 1998; Ault et al., 1998) have shown that ₁ receptor agonists inhibit NMDA-stimulated [³H]dopamine release from slices of rat and guinea pig striatum, prefrontal cortex, and nucleus accumbens. Concentrations of (+)-pentazocine below 1 µM, as well as the benzeneacetamide derivative BD737 (Bowen et al., 1992) seem to act via ₁ receptors to produce a concentration-related inhibition of release that was reversed by the selective ₁ antagonist DuP734, as well as by nonselective  receptor antagonists. We reasoned that if  receptors required an intact PKC signaling pathway, disruption of the pathway using PKC inhibitors should block the ability of  receptor agonists to regulate stimulated [³H]dopamine release. We have previously shown that ₂ receptors were linked to dopamine transporter function via activation of a Ca²⁺-dependent PKC isozyme (Derbez et al., 2002).

In the current study, we tested several inhibitors of PKC with differing profiles of selectivity for PKC isozymes, as well as an inhibitor of PLC, for their ability to affect ₁ receptor agonist-mediated regulation of NMDA-stimulated [³H]dopamine release from slices of rat striatum. Because of preliminary data showing that the pretreatment of tissue with the phorbol ester PMA, which acts at the diacyl glycerol (DAG) binding site on the PKC molecule, affected ₁ receptor regulation of release, we concentrated our investigations on the DAG-sensitive isozymes, the conventional PKC (cPKC) and novel PKC (nPKC) families. The atypical PKCs (aPKCs) are not sensitive to DAG and therefore are not affected directly by PMA. Our results support that PKC is a mediator of  receptor-mediated regulation of stimulated dopamine release and that the isozyme most likely to be involved is PKC.

	Materials and Methods

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Drugs and Reagents. The following chemicals were kindly provided by or obtained from the following sources: domperidone and nomifensine (Sigma/RBI, Natick, MA); L-ascorbic acid, chelerythrine (1,2-dimethoxy-12-methyl-[1,3]benzodioxolo[5,6-c]phenanthridinium chloride), GF109203x, and PMA (Sigma-Aldrich, St. Louis, MO); U73122 (BIOMOL Research Laboratories, Plymouth Meeting, PA); LY379196 (Lilly Research Laboratories, Indianapolis, IN); [³H]dopamine (Amersham Biosciences Inc., Piscataway, NJ); and (+)-pentazocine (Research Technology Branch, National Institute on Drug Abuse, Rockville, MD).

Measurement of Stimulated [³H]Dopamine Release from Striatal Slices. All experiments were carried out in accordance with the guidelines and the approval of the George Washington University Institutional Animal Use and Care Committee. Male Sprague-Dawley rats (Hilltop Laboratory Animals, Inc., Scottdale, PA) weighing 200 to 225 g were sacrificed by decapitation, and brains removed to ice. Striata were dissected, chopped in two planes at right angles into 250 × 250-µm strips with a Sorvall T-2 tissue sectioner, and suspended in modified Krebs-HEPES buffer (MKB: 127 mM NaCl, 5 mM KCl, 1.3 mM NaH₂PO₄, 2.5 mM CaCl₂, 15 mM HEPES, and 10 mM glucose, pH adjusted to 7.4 with NaOH) by titration through a plastic pipette. Buffers were oxygenated throughout the experiments and brain slices were kept at a constant temperature of 37°C. After three washes in MKB, tissue was resuspended in 20 ml of MKB and incubated for 30 min with 0.1 mM ascorbic acid and 15 nM [³H]dopamine. Tissue was then washed twice with 20 ml of MKB and once in 20 ml of MKB containing 10 µM nomifensine and 1 µM domperidone. These drugs were included in all subsequent steps to prevent reuptake of and feedback inhibition by the released [³H]dopamine. Tissue was suspended a final time in 7.5 ml of MKB, containing 10 µM nomifensine and 1 µM domperidone, and distributed in 275-µl aliquots between glass fiber discs into chambers of a superfusion apparatus (Brandel Inc., Gaithersburg, MD). MKB was superfused over tissue at a rate of 0.6 ml/min. Each experimental treatment was performed in triplicate within an individual, independent experiment (N). A low stable baseline release of approximately 0.9%/min was established over a 30-min period. Tissue was then stimulated by a 2-min exposure to 25 µM NMDA (S1). Inflow was then returned to nonstimulating buffer during a 10-min interstimulus interval (ISI). If (+)-pentazocine was being tested it was included at this time. Tissue was then exposed to a second stimulus (S2) identical to the first except in the presence of (+)-pentazocine. If a PKC or PLC inhibitor was being tested it was present throughout S1, ISI, and S2. In the experiments testing PMA, the drug was included for 30 min before S1. In experiments comparing long-term exposure versus short-term exposure to ₁ receptor agonist, (+)-pentazocine was included in ISI and S2, whereas in the latter (+)-pentazocine was only included in S2. The concentration of (+)-pentazocine used was 300 nM. This concentration of (+)-pentazocine should fully occupy ₁ receptors (K_i at ₁ = 5 nM; Vilner and Bowen, 1992), with approximately 16% occupancy at ₂ receptors (K_i = 1.5 µM; Vilner and Bowen, 1992). We have previously shown that 300 nM is on a plateau of the biphasic inhibition curve produced by increasing concentrations of (+)-pentazocine tested against NMDA-stimulated [³H]dopamine release (Gonzalez-Alvear and Werling, 1994). Inflow was once again returned to nonstimulating buffer before extraction of the remaining radioactivity in the tissue by a 45-min exposure to 0.2 N HCl at a reduced flow rate. Superfusates were collected at 2-min intervals in scintillation vials. Radioactivity remaining in tissue and the glass fiber filter discs was collected into the final vials. Radioactivity in samples was determined by liquid scintillation spectroscopy.

	Results

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To evaluate the involvement of PKC in ₁ receptor-mediated regulation of vesicular release of [³H]dopamine stimulated by 25 µM NMDA, we tested the PKC inhibitors chelerythrine, GF109203x, and LY379196 for their effects on NMDA-stimulated [³H]dopamine release or inhibition of release by (+)-pentazocine. We first tested single concentrations of PKC or PLC inhibitors to determine whether any PKC pathway might be involved in  receptor-mediated regulation of dopamine release. We tested the PLC inhibitor U731222 at 10 µM, the partially selective cPKC inhibitor GF109203x at 100 nM, the general PKC inhibitor chelerythrine at 5 µM, and the PKC-selective inhibitor LY379196 at 30 nM. Each was tested on both control NMDA-stimulated release and against (+)-pentazocine-mediated inhibition of NMDA-stimulated release. As shown in Table 1, there was no significant difference in the fractional release of [³H]dopamine stimulated by NMDA alone compared with the amount of [³H]dopamine release stimulated by NMDA in the presence of PMA, PKC inhibitors, or the PLC inhibitor. In contrast, all drugs tested were found to affect the ability of (+)-pentazocine to regulate release.

We then constructed a full concentration-response curve for each of the inhibitors. As seen in Fig. 1A, chelerythrine reversed the inhibition of NMDA-stimulated dopamine release by 300 nM (+)-pentazocine in a concentration-related manner. Chelerythrine has an overall K_i value at PKC of 660 nM undifferentiated for subtype (Way et al., 2000). The IC₅₀ value for chelerythrine at reversing the inhibition was similar to this value, lying between 300 nM and 1 µM. The highest concentrations of chelerythrine not only reversed completely the (+)-pentazocine-mediated inhibition but also produced a slight increase above control.

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Dose-response curves for the general PKC inhibitor chelerythrine (A), the cPKC selective inhibitor GF109203x (B), and the PKC-selective inhibitor LY379196 (C) on NMDA-stimulated [3H]dopamine release. Release of [3H]dopamine from slices of striatal tissue was stimulated by 25 µM NMDA or 25 µM NMDA in the presence of 300 nM (+)-pentazocine as indicated. Data are expressed as percentage of control NMDA-stimulated release. *, significantly different from NMDA alone at p < 0.05. n  three independent experiments. #, significantly different from 300 nM (+)-pentazocine without PKC inhibitor at p < 0.05. n  three independent experiments. Note break in y-axis.

Next, we tested GF109203x, a PKC inhibitor that is somewhat selective for the cPKCs (PKC, , and  isozymes) (K_i at  = 8.4 nM, I = 18 nM, II = 16 nM, and  = 20 nM), with affinities at PKC of 210 nM, and at PKC of 130 nM (Goekjian and Jirousek, 1999; Way et al., 2000). Its affinity at PKC is much lower (K_i = 5.8 µM). The IC₅₀ value for reversal of the (+)-pentazocine-mediated inhibition was between 10 and 30 nM (Fig. 1B). As seen with chelerythrine, GF109203x also at the higher concentrations increased dopamine release above control. At 10 nM GF109203x, release was at control levels. Fractional occupancy predicts that at 10 nM GF109203x, the PKCs significantly inhibited would have included PKC (54% blocked), I and II (37% blocked), and  (33% blocked), with <8% of other PKC isozymes blocked.

Last, we tested the selective PKC inhibitor LY379196. LY379196 has K_i values at I and II isozymes of 18 and 16 nM, and all other isozymes, including PKC, PKC, PKC, PKC, and PKC >300 nM (Dr. Louis Vignati, personal communication). There was full reversal even at very low concentrations of LY379196, with an IC₅₀ value of <10 nM. At this concentration, only the  isozymes should have been significantly occupied, with 2% of other identified PKCs blocked (Fig. 1C). Interestingly, there was no enhancement over control values with LY379196. This may suggest the enhancement over control shown by the other inhibitors is due to antagonism of some endogenous tone involving PKC isozymes that are not the  isoforms.

We also established dose-response curves against 300 nM (+)-pentazocine for the PLC inhibitor U73122. In Fig. 2, it can be seen that U73122 fully reversed (+)-pentazocine-mediated inhibition at a 3 µM concentration, and again produced an increase over control-stimulated release at higher concentrations. The IC₅₀ value was approximately 1 to 3 µM if the entire curve is considered.

View larger version (40K): [in this window] [in a new window]   Fig. 2.   Dose-response curve for the PLC inhibitor U73l22 on NMDA-stimulated [3H]dopamine release. Release of [3H]dopamine from slices of striatal tissue was stimulated by 25 µM NMDA or 25 µM NMDA in the presence of 300 nM (+)-pentazocine as indicated. Data are expressed as percentage of control NMDA-stimulated release. *, significantly different from NMDA alone at p < 0.05. N  3 independent experiments. #, significantly different from 300 nM (+)-pentazocine without U73122 at p < 0.05. n  three independent experiments. Note break in y-axis.

If the ₁ receptor requires an intact PKC system to exert its regulation on release of [³H]dopamine, we hypothesized that prior exposure to the phorbol ester PMA should prevent regulation of [³H]dopamine release by (+)-pentazocine. In this set of experiments, tissue was exposed to 1 µM PMA during the 30-min equilibration phase (see Materials and Methods) before the collection of superfusate fractions. As seen in Fig. 3, prior pretreatment with 1 µM PMA completely blocked the inhibition of release by 300 nM (+)-pentazocine, supporting the hypothesis that intact, active PKC is required for ₁ receptor-mediated regulation of release. Another observation that supports this hypothesis is depicted in Fig. 4. In these experiments, tissue was exposed to (+)-pentazocine at the indicated concentration during S2 only instead of the usual exposure during the 10-min ISI interval preceding the S2. Inclusion of (+)-pentazocine in S2 only did not produce any inhibition. However, with the standard protocol of a 12-min exposure (i.e., in ISI and S2), inhibition by (+)-pentazocine was observed as reported previously.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Effects of PMA on NMDA-stimulated [3H]dopamine release. Release of [3H]dopamine from slices of striatal tissue was stimulated by 25 µM NMDA (open columns) or 25 µM NMDA in the presence of 300 nM (+)-pentazocine (filled columns). Experiments were carried out with (+PMA) or without (PMA) a prior pretreatment of 1 µM PMA 30 min before the first stimulus (S1). Data are expressed as percentage of NMDA-stimulated release. *, significantly different from NMDA alone at p < 0.05. There was no significant difference in NMDA-stimulated release with PMA pretreatment (S2/S1 = 0.56 ± 0.03) compared with NMDA-stimulated release without PMA pretreatment (S2/S1 = 0.54 ± 0.04). n  three independent experiments. Note break in y-axis.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Dose-response curves for (+)-pentazocine on NMDA-stimulated [3H]dopamine release. Curves compare shorter exposure versus longer exposure to  agonist. (+)-Pentazocine was included either during the 10-min ISI and the S2 (bottom curve), or only during S2 (top curve). Data are expressed as percentage of control NMDA-stimulated release. Note break in y-axis. There is a significant difference between the two curves as determined by t test at p < 0.05. n = three independent experiments.

	Discussion

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The signaling mechanisms used by  receptors have been largely uncharacterized. Early reports on association of G proteins with  receptors were conflicting, with some investigators showing an association (Itzhak, 1989; Tokuyama et al., 1999; Hayashi et al., 2000; Meyer et al., 2002) and others refuting those assertions (DeHaven-Hudkins et al., 1992). The cloned  receptor protein is too small to be a classic G protein-coupled receptor, and no recognition site is apparent from sequence analysis for the direct interaction of the ₁ receptor with G proteins.

Ca²⁺ homeostasis has been shown to be modified by  receptor activation. Hayashi et al. (2000) demonstrated that in NG108 cells, the  receptor agonist (+)-pentazocine as well as pregnenolone sulfate affect intracellular Ca²⁺ levels by releasing Ca²⁺ from the ER, and that the mobilization of Ca²⁺ was blocked by the ₁ receptor antagonist NE-100. Hayashi and Su (2001) further demonstrated that the interaction of  receptors with the IP₃ binding site on the ER caused a dissociation of ankyrin B (ANK220) from ER. ANK220 is a member of a family of cytoskeletal adapter proteins that interconnect membrane proteins with the cell cytoskeleton (Bennett and Stenbuck, 1979) and is present on ER, PM, and Golgi complex (De Matteis and Morrow, 1998; Tuvia et al., 1999). Ankyrin has been identified as a regulator of Ca²⁺ signaling at the IP₃ site on the ER (Bourguignon and Jin, 1995). Additionally, Brent et al. (1997) have shown that the nonsubtype-selective  ligand 1,3-di(2-totyl)guanidine, as well as (+)-pentazocine, increased basal phosphorylation of synapsin Ib and dynamin, and decreased depolarization-stimulated phosphorylation of synapsin Ib in synaptosomes prepared from rat forebrain. Novakova et al. (1998) have reported that treatment of rat cardiac myocytes with the ₁-selective agonist BD737 potentiated electrically evoked amplitudes of contraction and Ca²⁺ transients as well as an increase in spontaneous twitch. These effects were blocked by prior depletion of IP₃-sensitive Ca²⁺ stores with thapsigargin, and also by the PLC inhibitor neomycin, and were reversed by a  receptor antagonist. BD737 alone also increased the intracellular IP₃ concentration.

The relationship of  receptor activation to Ca²⁺ changes and IP₃ turnover, as well as evidence from our own laboratory demonstrating the abolition of ₂ receptor-mediated regulation of dopamine transporter activity by the PKC inhibitor GF109203x (Derbez et al., 2002) suggests that the PKC signaling system could participate in ₁ receptor-mediated regulation of vesicular dopamine release. PKC has been implicated in enhancement of release of other neurotransmitters (Majewski and Iannazzo, 1998), including adenosine and norepinephrine. PKC activation has also been linked to inhibition of NMDA-stimulated Ca²⁺ changes in cerebellar granule cells (Snell et al., 1994).

The PKC family includes at least 12 isozymes of threonine/serine kinases that respond to specific stimuli (for review, see Way et al., 2000). There are three major groups of PKCs. The first of these groups is the cPKCs, which are the , I, II, and  types. The cPKCs are Ca²⁺-dependent and activated by phosphatidyl serine (PS) and DAG. Members of a second group, the nPKCs , , , and , are Ca²⁺-independent, but like the cPKCs are regulated by PS and DAG. Finally, the aPKCs  and / are Ca²⁺-independent, do not require DAG, but are regulated by PS. Localization to various tissues also varies by isoform. PKC and  are found in central nervous system. In brain, it seems the types of PKC involved in transmitter release include the cPKCs and the nPKCs, but not the aPKCs. PKC, PKC, and PKC are the isozymes localized to synaptosomes in rat hippocampus, cortex, and cerebellum. In rat cerebellar cortex synaptosomes, when PKC and PKC are down-regulated, without down-regulation of PKC, the facilitative effects of PKC activators on norepinephrine release are abolished, suggesting that PKC is not involved in norepinephrine release (Oda et al., 1991). Because the isozymes present in hippocampal synaptosomes are PKC, , and , one might conclude that the isozyme involved in norepinephrine release is either  or .

PKC is activated when a stimulus at a receptor initiates dissociation of the subunits of Gq associated with phospholipase C, causing the cleavage of IP₃ and DAG from membrane-bound phospholipids. The DAG activates PKC, whereas the released IP₃ can act at its own receptor on the ER to release Ca²⁺. Arachidonic acid and lysophosphatidyl choline can also activate PKC and may have a synergistic action with DAG on PKC activity (Schachter et al., 1996). After activation, PKC translocates to and phosphorylates its target substrate(s).

We sought to determine which isozymes were likely to be involved in  receptor regulation of dopamine release. Although the selectivity of available PKC inhibitors for the various isozymes is not ideal, a tentative identification of likely candidate isozymes can be made by determining which inhibitors affect ₁ receptor regulation, and by comparing their overlap in ability to inhibit the various isozymes. Our observations in the current study that a general inhibitor (chelerythrine), the partially selective cPKC inhibitor GF109203x, acting with an IC₅₀ value compatible with its K_i value at the cPKCs, and a PKC-selective inhibitor (LY379176) at low nanomolar concentrations all abolish (+)-pentazocine-mediated regulation of stimulated [³H]dopamine release suggest the isozyme involved is likely to be one of the  isoforms. Further support for either cPKC or nPKC involvement is the observation that prior treatment with PMA abolishes regulation of release by (+)-pentazocine. PMA binds at the DAG site, which is absent from the aPKCs. Although PMA has many cellular targets, its ability to abolish (+)-pentazocine's effects, coupled with results in the PKC inhibitor experiments, is consistent with PMA's actions at down-regulating PKC in the current study.

Other studies support an involvement of PKC isozymes in ₁ receptor action. Using polyclonal antibodies generated against the ₁ binding protein from guinea pig liver, Morin-Surun et al. (1999) have shown that when stimulated by exposure to (+)-pentazocine, both the ₁ receptor and the I and II isoforms of PKC in guinea pig hypoglossal neurons translocate from a diffuse location in the cytosol to the PM. The translocation was temporally associated with a desensitization of the (+)-pentazocine-induced inhibition of firing of hypoglossal neurons. These finding are consistent with our preliminary data suggesting the isoform of PKC involved in ₁ receptor regulation of exocytotic dopamine release could be PKC. The demonstration that the ₁ receptor translocates may explain why it has been identified in multiple PM and subcellular locations (McCann et al., 1994; Alonso et al., 2000). It also helps one envision the  receptor as a different receptor from the traditional PM receptor for many other transmitters. Because there is now evidence that ₁ receptors and PKC translocate when stimulated, it is possible, although not necessary, that ₁ receptors and PKC directly interact.

Exactly how ₁ receptor activation affects the PKC signaling pathway is not addressed by the current study. The fact that there must be a sufficient exposure to the ₁ agonist (+)-pentazocine as shown in Fig. 4 might indicate that ₁ receptor agonist causes modulation of PKC activity. The Morin-Surun study on the (+)-pentazocine-mediated effect of regulation of firing in guinea pig hypoglossal neurons suggests that desensitization to (+)-pentazocine occurs. However, this possibility of desensitization and/or down-regulation in our system remains to be confirmed. From the current data, it can only be said that an intact PKC is required for  receptor activation to regulate stimulated [³H]dopamine release.

In summary, we have shown that our previously reported ₁ agonist-mediated inhibition of NMDA-stimulated [³H]dopamine release is transduced by a PKC signaling system most likely to involve the PKC isozyme. Regulation is abolished by pretreatment with PMA, PKC, or PLC inhibitors, and requires sufficient exposure time to  agonist. We believe this to be the first report of ₁ receptor signaling being associated with PKC in regulation of transmitter release.