Effect of Novel -Conotoxins on Nicotine-Stimulated [H]Dopamine Release from Rat Striatal Synaptosomes

Nicotine’s action on the midbrain dopaminergic neurons is mediated by nicotinic acetylcholine receptors (nAChRs) that are present on the cell bodies and the terminals of these neurons. Previously, it was suggested that one of the nAChR subtypes located on striatal dopaminergic terminals may be an 3 2 subtype, based on partial inhibition of nicotine-stimulated [H]dopamine release by -conotoxin MII, a potent inhibitor of heterologously expressed 3 2 nAChRs. More recent studies indicated that -conotoxin MII also potently blocks 6-containing nAChRs. In the present study, we have examined the nAChR subtype(s) modulating [H]dopamine release from striatal terminals by using novel -conotoxins that have 37to 78-fold higher selectivity for 6versus 3-containing nAChRs. All of the peptides partially (20–35%) inhibit nicotine-stimulated [H]dopamine release with IC50 values consistent with those obtained with heterologously expressed rat 6-containing nicotinic acetylcholine receptors. These results, together with previous studies by others, further support the idea that 6-containing nicotinic receptors modulate nicotine-stimulated dopamine release from rat striatal synaptosomes. Nicotinic acetylcholine receptors (nAChRs) are members of the large family of ligand-gated ion channels. Neuronal nAChRs have a pentameric structure composed of and subunits (Anand et al., 1991; Cooper et al., 1991). Through molecular cloning, six mammalian neuronal ( 2– 7) and three ( 2– 4) subunits have been identified in the brain (Sargent, 1993; McGehee and Role, 1995). Heteromeric receptors are composed of different combination of these subunits. Heterologous expression systems have revealed that and subunits both contribute to the diversity in biochemical and physiological properties of this class of receptors (Gross et al., 1991; Luetje and Patrick, 1991; Cachelin and Rust, 1995; McGehee and Role, 1995). Nigrostriatal dopamine (DA) neurons, which originate from within the substantia nigra compacta and send projections to the dorsal striatum, are involved in voluntary motor control. The selective loss of these neurons has been implicated in the pathology of the motor deficits associated with Parkinson’s disease. Nicotine’s actions on these neurons, and subsequent dopamine release within the striatum, may partly underlie this alkaloid’s potential therapeutic effects in patients with Parkinson’s. Midbrain (DA) neurons possess nicotine binding sites (Clarke and Pert, 1985), and nicotine directly activates these neurons (Pidoplichko et al., 1997; Yin and French, 2000). Moreover, nicotine stimulates release of DA within the striatal target region by direct action on nicotinic receptors on the DAergic terminals (Rapier et al., 1990; Grady et al., 1992; Clarke and Reuben, 1996; Wonnacott, 1997). In situ hybridization and immunohistochemical studies have revealed the presence of a variety of nAChR subunit mRNAs and protein within midbrain DAergic neurons, including 3– 7 and 2 and 3 (Charpantier et al., 1998; Elliott et al., 1998; Sorenson et al., 1998; Klink et al., 2001; Azam et al., 2002), that could potentially result in numerous distinct subtypes of nAChRs. Therefore, due to lack of subtype-specific ligands, it has been difficult to determine the exact subtypes of nAChRs mediating DA neurotransmission. Recently, it was shown that -conotoxin MII ( -MII), a Conus toxin isolated from Conus magus that potently inhibits the 3 2 nAChR subtype (Cartier et al., 1996), inhibited approximately 30 to 40% of nicotine-stimulated [H]DA release from striatal synaptosomes (Kulak et al., 1997; Kaiser et al., 1998; Grady et al., 2001). However, more recent data indicate that -MII also has high affinity for 6-containing receptors. Studies with mutant animals have shown that I-MII binding is largely preserved in 3 knockout mice, but it is abolished in 6 knockout mice (Champtiaux et al., 2002; This work was supported by National Institute of Health Grant MH53631 (to J.M.M.) and Ruth L. Kirschstein National Research Service Award Postdoctoral Fellowship DA016835 (to L.A.). Portions of this work have been presented previously in Azam L, Dowell C, and McIntosh JM (2002) Characterization of nicotine-mediated dopamine release from striatal synaptosomes using a novel -conotoxin. Program 242.9. 2002 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2002. Online. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.104.071456. ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; DA, dopamine; -MII, -conotoxin MII; -PIA, -conotoxin PIA. 0022-3565/05/3121-231–237$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 312, No. 1 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 71456/1181223 JPET 312:231–237, 2005 Printed in U.S.A. 231 at A PE T Jornals on A ril 9, 2017 jpet.asjournals.org D ow nladed from Whiteaker et al., 2002). These studies suggest that the primary target of -MII in DA neurons may be an 6rather than 3-containing receptor. Until now, no ligands that distinguish between 6* and 3* nAChRs (asterisk indicates the possible presence of other subunits) have been available. In the present study, we have characterized nicotine-modulated [H]DA release from rat striatal synaptosomes using the novel -conotoxin PIA ( PIA), isolated from Conus purpurascens that has approximately 78-fold higher selectivity for 6versus 3-containing receptors. In addition, three recently developed structural analogs of -MII, with 37-, 54-, and 75-fold selectivity for 6versus 3-containing receptors, were also used. The results implicate the involvement of 6* rather than 3* receptors in nicotine-evoked dopamine release from DAergic terminals. Materials and Methods Materials. The chemicals were obtained from the following sources: ( )-Nicotine hydrogen tartarate, pargyline HCl, bovine serum albumin, ascorbic acid (Sigma-Aldrich, St. Louis, MO), [H]dopamine (dihydroxyphenylethylamine 3,4 [7-H]; 28–30 Ci/mmol) (PerkinElmer Life and Analytical Sciences, Boston, MA), Ecolume scintillation cocktail (MP Biomedicals, Irvine, CA). -Conotoxins were synthesized as described previously (Cartier et al., 1996; McIntosh et al., 2004). Tissue Preparation. Adult male albino rats (Simonsen Laboratories, Gilroy, CA) were kept two per cage on a 12:12-h light/dark cycle, with food and water available ad libitum. For each experiment, four adult male rats between 60 and 90 days old were used. The rats were decapitated and brains quickly removed. This procedure was approved by the Institutional Animal Care and Use Committee and is consistent with Federal guidelines. Synaptosomes were prepared essentially as described by Kulak et al. (1997). Briefly, the striata were quickly dissected on ice and placed in ice-cold 0.32 M sucrose buffer, pH 7.4 to 7.5. The dissected striata were homogenized by 14 gentle up and down strokes, followed by centrifugation at 1000g for 10 min at 4°C. The supernatant was centrifuged at 12,000g for 20 min at 4°C. The resulting P2 pellet was resuspended in 2 ml of Krebs-HEPES buffer (superfusion buffer) with composition 128 mM NaCl, 2.4 mM KCl, 1.2 mM KH2PO4, 0.6 mM MgSO4, 3.2 mM CaCl2, 25 mM HEPES, and 10 mM glucose and supplemented with 1 mM ascorbic acid and 0.1 mM pargyline. The synaptosomes were incubated for 10 min at 37°C to equilibrate with the superfusion buffer, followed by another 10-min incubation with 0.12 M [H]dopamine (specific activity 28–30 Ci/mmol) at 37°C. The synaptosomes were centrifuged at 3500 rpm for 5 min to get rid of excess radiolabeled dopamine. The pellet was resuspended in 4 ml of superfusion buffer, and 1 ml was transferred into each of four conical tubes containing 3 ml of superfusion buffer and subsequently loaded into the superfusion chambers containing 13-mm-diameter A/E glass fiber filters (Gelman Instrument Co., Ann Arbor, MI). One tube (4-ml total volume) contained enough synaptosomes for six chambers of the superfusion apparatus. Superfusion. The superfusion system had 12 identical channels and was set up as described in Kulak et al. (1997). Once synaptosomes were loaded into the superfusion apparatus, they were washed for 20 min with either superfusion buffer alone or buffer plus varying concentrations of the toxins, at a rate of 0.5 ml/min. -Conotoxin MII[E11A], due to its slow on-rate, was flowed on for 40 min. After the wash period, 2-min fractions were collected in 5-ml polypropylene vials containing 4 ml of Ecolume scintillation cocktail. At the end of the third 2-min fraction, a 1-min pulse of nicotine or nicotine plus toxin was applied, followed by five 2-min washes with superfusion buffer alone. For studies where -MII and -PIA or -MII and -MII[E11A] were coapplied, synaptosomes were perfused for 20 or 40 min, respectively, with buffer containing 100 nM -MII and 100 nM -PIA or 100 nM -MII and 10 nM -MII[E11A] and pulsed with nicotine as described above. At the end of the superfusion, filters containing the synaptosomes were taken out and placed directly in vials containing 4 ml of Ecolume to determine total [H]DA uptake. Radioactivity collected in each fraction was quantitated by liquid scintillation spectroscopy, with Beckman Coulter 5801 and 9800 liquid scintillation counters, tritium efficiency approximately 50%. Data Analysis. Throughout this article, tritium release is presumed to correspond directly to amounts of radiolabeled transmitter release, because it has been shown previously that tritium released by nAChR agonists is proportional to total radiolabeled transmitter released (Rapier et al., 1988). Baseline release was determined as average of two fractions before and two fractions after the peak release. Average baseline was subtracted from the evoked release and the resulting values divided by the baseline to yield the evoked release as a percentage over baseline. For all data, except the release profiles in Fig. 1, percentage of release over baseline was normalized to average release with nicotine alone. IC50 values were determined by nonlinear regression analysis using Prism (Graphpad Software Inc., San Diego, CA). All statistical analysis was performed with Prism. Toxin effects were analyzed by one-way analysis of variance, followed by Dunnett’s post hoc for comparisons with nicotine control or Newman-Keuls or Bonferroni’s (where indicated) for multiple pairwise comparisons.