N,N'-Alkane-diyl-bis-3-picoliniums as nicotinic receptor antagonists: inhibition of nicotine-evoked dopamine release and hyperactivity

Abstract

The current study evaluated a new series of N,N'-alkane-diyl-bis-3-picolinium (bAPi) analogs with C6-C12 methylene linkers as nicotinic acetylcholine receptor (nAChR) antagonists, for nicotine-evoked [3H]dopamine (DA) overflow, for blood-brain barrier choline transporter affinity, and for attenuation of discriminative stimulus and locomotor stimulant effects of nicotine. bAPi analogs exhibited little affinity for alpha4beta2* (* indicates putative nAChR subtype assignment) and alpha7* high-affinity ligand binding sites and exhibited no inhibition of DA transporter function. With the exception of C6, all analogs inhibited nicotine-evoked [3H]DA overflow (IC50 = 2 nM-6 microM; Imax = 54-64%), with N,N'-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB; C12) being most potent. bPiDDB did not inhibit electrically evoked [3H]DA overflow, suggesting specific nAChR inhibitory effects and a lack of toxicity to DA neurons. Schild analysis suggested that bPiDDB interacts in an orthosteric manner at nAChRs mediating nicotine-evoked [3H]DA overflow. To determine whether bPiDDB interacts with alpha-conotoxin MII-sensitive alpha6beta2-containing nAChRs, slices were exposed concomitantly to maximally effective concentrations of bPiDDB (10 nM) and alpha-conotoxin MII (1 nM). Inhibition of nicotine-evoked [3H]DA overflow was not different with the combination compared with either antagonist alone, suggesting that bPiDDB interacts with alpha6beta2-containing nAChRs. C7, C8, C10, and C12 analogs exhibited high affinity for the blood-brain barrier choline transporter in vivo, suggesting brain bioavailability. Although none of the analogs altered the discriminative stimulus effect of nicotine, C8, C9, C10, and C12 analogs decreased nicotine-induced hyperactivity in nicotine-sensitized rats, without reducing spontaneous activity. Further development of nAChR antagonists that inhibit nicotine-evoked DA release and penetrate brain to antagonize DA-mediated locomotor stimulant effects of nicotine as novel treatments for nicotine addiction is warranted.

Fig. 1

Chemical structures, full chemical names and abbreviations of N,N′-alkane-diyl-bis-3-picolinium (bAPi) salts.

Fig. 2. At high concentrations, N,N′-alkane-diyl-bis-3-picolinium (bAPi) analogs inhibit [³H]nicotine binding (top panel) and [³H]MLA binding to rat brain membranes (bottom panel)

Analog abbreviations are provided in Fig. 1. Cytisine and nudicauline were used as a positive control for [³H]nicotine and [³H]MLA binding assays, respectively. Inhibition of the binding of 3 nM [³H]nicotine ([³H]NIC, top panel) or 2.5 nM [³H]MLA (bottom panel) was determined in separate series of experiments. Nonspecific binding was determined in the presence of 10 μM nicotine or cytisine for [³H]nicotine binding assays and 1 mM nicotine for [³H]MLA binding assays. Data are fmol/mg protein expressed as % of control. Control represents [³H]nicotine binding (85.2 ± 6.93 fmol/mg protein; mean ± S.E.M) and [³H]MLA binding (69.6 ± 9.72 fmol/mg protein; mean ± S.E.M), respectively, in the absence of analog; n= 3–5 rats/analog/binding assay. Curves were generated by nonlinear regression.

Fig. 3. N,N′-Alkane-diyl-bis-3-picolinium (bAPi) analogs inhibit nicotine-evoked [³H]DA overflow from rat striatal slices (Top panel)

Analog abbreviations are provided in Fig. 1. Assay buffer contained nomifensine (10 μM) and pargyline (10 μM) throughout superfusion. Slices were superfused in the absence (control) or presence of analog for 60 min prior to nicotine addition to the buffer; superfusion continued for 60 min with nicotine added to the buffer. Control represents [³H]DA overflow in response to 10 μM nicotine (3.12 ± 0.16 total [³H]DA overflow as a percent of tissue [³H]content, mean ± S.E.M.). Data are expressed as % of nicotine control; n = 5–10 rats/analog. Curves for concentration response were generated by nonlinear regression. Time course of bPiDDB-induced inhibition of nicotine-evoked [³H]DA overflow is shown in the bottom panel. Time course data were used to generate [³H]DA overflow data for bPiDDB, shown in the top panel. Slices were superfused with a range of concentrations (1 nM–10 μM) of bPiDDB for 60 min. After collection of the third sample, nicotine (10 μM) was added to the superfusion buffer in the absence (control) and presence of bPiDDB and superfusion continued for 60 min. In each experiment, a control slice was superfused in the absence of bPiDDB and presence of nicotine to determine nicotine-evoked total [³H]DA overflow. The arrow indicates the time point at which nicotine was added to the superfusion buffer. Data are expressed as fractional release as a percent of basal (mean ± S.E.M.), n = 6 rats. Basal was 0.94 ± 0.04 fractional release as % tissue [³H]content.

Fig. 4. Schild analysis for bPiDDB inhibition of nicotine-evoked [³H]DA overflow from superfused rat striatal slices

Assay buffer contained nomifensine (10 μM) and pargyline (10 μM) throughout superfusion. After collection of the third sample, slices were superfused with buffer in the absence and presence of bPiDDB (1, 3 or 10 nM) for 60 min prior to the addition of nicotine (0.1 nM–100 μM) to the buffer, and superfusion continued for an additional 60 min. For each nicotine concentration, the control response is that for nicotine in the absence bPiDDB. Data are presented as mean ± S.E.M. of total [³H]overflow during the 60 min exposure to nicotine in the absence and presence of bPiDDB; n = 5 rats. Concentration response curves were generated by nonlinear regression. Fig. 4 inset shows the Schild regression in which the log of dr −1 was plotted as a function of log of bPiDDB concentration. Data were fit by linear regression.

Fig. 5. α-Conotoxin MII inhibition of nicotine (10 μM)-evoked [³H]DA overflow from superfused rat striatal slices is concentration-dependent, but incomplete

The concentration was determined at which α-conotoxin (α-CtxMII) produced maximal inhibition of nicotine-evoked [³H]DA release from rat striatal slices using the current experimental conditions. Assay buffer contained nomifensine (10 μM) and pargyline (10 μM) throughout superfusion. Slices were superfused in the absence (control) or presence of α-CtxMII (0.1 – 30 nM) for 36 min, and superfusion continued for 36 min following addition of nicotine to the buffer. Control represents [³H]DA overflow in response to 10 μM nicotine (1.54 ± 0.18 total [³H]DA overflow as a percent of tissue [³H]content, mean ± S.E.M.). Data are expressed as mean ± S.E.M. of % of nicotine control for n=6 rats and the concentration-response curve was generated using nonlinear regression.

Fig. 6. Concomitant exposure to concentrations of bPiDDB and α-CtxMII produces inhibition of nicotine-evoked [³H]DA overflow not different from that following exposure to either antagonist alone

Superfusion buffer contained nomifensine (10 μM) and pargyline (10 μM). Maximal inhibitory concentrations of α-CtxMII (1 nM) and bPiDDB (10 nM) were selected from previous concentration response analysis. After collection of the second sample, slices were superfused in the absence or presence of α-CtxMII, bPiDDB or α-CtxMII + bPiDDB for 36 min. Superfusion continued for 36 minutes following addition of nicotine (10 μM) to the buffer. Control represents nicotine-evoked [³H]DA overflow in the absence of antagonist (1.99 ± 0.52 total [³H]DA overflow as a percent of tissue [³H]content, mean ± S.E.M.). Data are expressed as mean ± S.E.M. of % control. * indicates different from control (p < 0.01). n = 8 rats. Inset: Time course of nicotine-evoked [³H]DA overflow in the absence and presence of α-CtxMII, bPiDDB or α-CtxMII + bPiDDB. Data are expressed as fractional release as a percent of basal outflow. Arrow indicates the time point at which nicotine was added to the superfusion buffer.

Fig. 7. N,N′-Alkane-diyl-bis-3-picolinium (bAPi) analogs do not inhibit [³H]DA uptake into rat striatal synaptosomes

Analog abbreviations are provided in Fig. 1. GBR 12909 was used as a positive control for these experiments. Data are pmol/min/mg of specific [³H]DA uptake expressed as % of control; n= 3–4 rats. Control represents [³H]DA uptake in the absence of analog (33.6 ± 1.75 pmol/min/mg protein; mean ± S.E.M.). Nonspecific [³H]DA uptake was determined in the presence of 10 μM nomifensine. Curves were generated using nonlinear regression; analog abbreviations are provided in Fig. 1.

Fig. 8. Reduction of cortical BBB [³H]choline permeability (PS) in the presence of N,N′-alkane-diyl-bis-3-picolinium (bAPi) analogs (250 μM) using the in-situ rat brain perfusion technique, suggesting interaction with the BBB choline transporter

Data represent the mean (± SEM) for 3–6 independent observations (***p < 0.001).

Fig. 9. In contrast to mecamylamine and DHβE, the bAPi analogs do not inhibit the discriminative stimulus effect of nicotine

Discriminative stimulus and response rate effects of nicotine in rats (n=6) trained to discriminate nicotine (0.2 mg/kg, sc) from saline (panels a and b), and in nicotine-trained rats pretreated with either mecamylamine, DHβE (panels c and d), or bAPi analog (panels e and f). Analog abbreviations are provided in Fig. 1. Data points represent the mean (± SEM) percentage of total responses occurring on the nicotine-appropriate lever (upper panels) and rate of responding (lower panels) as a function of nicotine (panels a and b) or pretreatment (panels c–f) dose. Asterisks indicate a significant difference relative to saline (S) control (*p < 0.05, **p < 0.01).

Fig. 10. Mecamylamine and DHβE inhibit the locomotor stimulant effect of nicotine

Mecamylamine (MEC; 0.1–3.0 mg/kg, sc; left panel) or DHβE (0.1–3.0 mg/kg, sc; right panel) pretreatment was given prior to nicotine (0.4 mg/kg) administration in nicotine-sensitized rats. Points represent the mean (± SEM) total distance traveled (cm) as a function of pretreatment dose. Asterisks indicate a significant difference from the corresponding saline (S) control (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 11. bAPi analogs differentially inhibit the locomotor stimulant effects of nicotine

bAPi analogs (0.58–5.8 μmoles/kg, sc) were given prior to nicotine (0.4 mg/kg) or saline administration in nicotine-sensitized rats. Analog abbreviations are provided in Fig. 1. Points represent the mean (± SEM) total distance traveled (cm) as a function of pretreatment dose. Asterisks indicate a significant difference from corresponding saline (S) control (*p < 0.05, **p < 0.01, ***p < 0.001).