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Tobacco products are some of the most commonly used psychoactive drugs worldwide. Besides nicotine, alkaloids in tobacco include cotinine, myosmine, and anatabine. Scientific investigation of these constituents and their contribution to tobacco dependence is less well developed than for nicotine. The present study evaluated the nucleus accumbens dopamine-releasing properties and rewarding and/or aversive properties of nicotine (0.2-0.8 mg/kg), cotinine (0.5-5.0 mg/kg), anatabine (0.5-5.0 mg/kg), and myosmine (5.0-20.0 mg/kg) through in vivo microdialysis and place conditioning, respectively, in adult and adolescent male rats. Nicotine increased dopamine release at both ages, and anatabine and myosmine increased dopamine release in adults, but not adolescents. The dopamine release results were not related to place conditioning, as nicotine and cotinine had no effect on place conditioning, whereas anatabine and myosmine produced aversion in both ages. While the nucleus accumbens shell is hypothesized to play a role in strengthening drug-context associations following initiation of drug use, it may have little involvement in the motivational effects of tobacco constituents once these associations have been acquired. Effects of myosmine and anatabine on dopamine release may require a fully developed dopamine system, since no effects of these tobacco alkaloids were observed during adolescence. In summary, while anatabine and myosmine-induced dopamine release in nucleus accumbens may play a role in tobacco dependence in adults, the nature of that role remains to be elucidated.

Chronic pain is inadequately managed with currently available classes of analgesic drugs. Recently, peptide antagonists of the α9α10 nicotinic acetylcholine receptor were shown to be analgesic. The present study was conducted to characterize a novel small molecule, non-peptide antagonist at nicotinic receptors. The tetrakis-quaternary ammonium compound ZZ-204G was evaluated for functional activity on cloned nicotinic receptors expressed in Xenopus oocytes. In-vivo efficacy was assessed in rat models of tonic inflammatory pain (formalin test), neuropathic pain (chronic constriction nerve injury), and thermal nociception (tail flick test). ZZ-204G was an antagonist at nicotinic receptors inhibiting the α9α10 subtype with an IC₅₀ of 0.51 (0.35-0.72) nM. Antagonist activity at other nicotinic subtypes (α1β1δe, α2β2, α2β4, α3β2, α3β4, α4β2, α4β4, α6/α3β2β3, α6/α3β4 and α7) was 10-1000-fold lower than at the α9α10 subtype. In competition binding assays, the k(i) of ZZ-204G at γ-aminobutyric acid(A), serotonin(3), γ-aminobutyric acid(B), κ- and μ-opioid receptors was 1000- to >10,000-fold lower than at α9α10 nicotinic receptors. Parenteral administration of ZZ-204G dose-dependently decreased nociceptive behaviors (paw flinches) in the formalin test and mechanical hyperalgesia in the chronic constriction nerve injury model of neuropathic pain. ZZ-204G was not antinociceptive in the tail flick assay. Results from the rotarod assay indicated that lower doses of ZZ-204G that were analgesic did not alter motor function. In summary, ZZ-204G represents a prototype small molecule antagonist for α9α10 nicotinic receptors and provides a novel molecular scaffold for analgesic agents with the potential to treat chronic inflammatory or neuropathic pain.

Although several therapeutic agents are available to aid in tobacco smoking cessation, relapse rates continue to be high, warranting the development of alternative pharmacotherapies. Nicotine-evoked dopamine release from its presynaptic terminals in the central nervous system leads to reward which maintains continued tobacco use. The ability of indolizidine (-)-235B' and a sub-library of structurally related analogs to inhibit nicotine-evoked [(3)H]dopamine release from rat striatal slices was determined in the current study. Indolizidine (-)-235B' inhibited nicotine-evoked [(3)H]dopamine release in a concentration-dependent manner (IC(50)=42 nM, I(max)=55%). Compound (-)-237D, the double bond-reduced analog, afforded the greatest inhibitory potency (IC(50)=0.18 nM, I(max)=76%), and was 233-fold more potent than indolizidine (-)-235B'. The des-8-methyl aza-analog of indolizidine (-)-235B', ZZ-272, also inhibited nicotine-evoked [(3)H]dopamine release (IC(50)=413 nM, I(max)=59%). Concomitant exposure to maximally effective concentrations of indolizidine (-)-235B', ZZ-272 or (-)-237D with a maximally effective concentration of α-conotoxin MII, a selective antagonist for α6β2-containing nicotinic receptors, resulted in inhibition of nicotine-evoked [(3)H]dopamine release no greater than that produced by each compound alone. The latter results suggest that indolizidine (-)-235B', (-)-237D, ZZ-272 and α-conotoxin MII inhibit the same α-conotoxin MII-sensitive nicotinic receptor subtypes. Thus, indolizidine (-)-235B' and its analogs act as antagonists of α6β2-nicotinic receptors and constitute a novel structural scaffold for the discovery of pharmacotherapies for smoking cessation.

Lobelane, a minor alkaloid of Lobelia inflata and a synthetic, des-oxy analog of lobeline, has good affinity for the vesicular monoamine transporter and the dopamine transporter. The current study examined the ability of lobelane to specifically decrease methamphetamine self-administration. Rats were trained on a fixed ratio 5 schedule of reinforcement to self-administer methamphetamine (0.05 mg/kg/infusion, i.v.) or to respond for sucrose pellets. Upon reaching stable responding, rats were pretreated with lobelane (0.1, 1, 3, 5.6, or 10 mg/kg, s.c.) or saline, 15 min prior to the operant session. To assess the effect of repeated lobelane on methamphetamine self-administration, rats were pretreated with lobelane (5.6 or 10 mg/kg, s.c.) for 7 sessions. Behavioral specificity was further investigated by assessing the effects of lobelane (0.1, 1, 3, 5, or 10 mg/kg, s.c.) or saline on locomotor activity. Within the dose range tested, lobelane dose-dependently decreased methamphetamine self-administration, while having no effect on sucrose-maintained responding. Locomotor activity was decreased following only the highest dose of lobelane (10 mg/kg). Across repeated pretreatments, tolerance developed to the effect of lobelane on methamphetamine self-administration, demonstrating that the ability of lobelane to specifically decrease methamphetamine self-administration is a transient effect. Thus, taken together, the results show that although lobelane interacts with the pharmacological targets believed to be responsible for its ability to decrease methamphetamine self-administration, removal of the oxygen functionalities from the lobeline molecule may have afforded a compound with an altered pharmacokinetic and/or pharmacodynamic profile.

In previous in vivo voltammetry studies, acute nicotine administration increased striatal dopamine clearance. The current study aimed to determine whether nicotine also increases [(3)H]dopamine uptake across the time course of the previous voltammetry studies and whether dopamine transporter trafficking to the cell surface mediates the nicotine-induced augmentation of dopamine clearance in striatum. Rats were administered nicotine (0.32 mg/kg, s.c.); striatal synaptosomes were obtained 5, 10, 40 or 60 min later. Nicotine increased (25%) the V(max) of [(3)H]dopamine uptake at 10 and 40 min. To determine whether the increase in V(max) was due to an increase in dopamine transporter density, [(3)H]GBR 12935 (1-(2-[bis(4-fluorophenyl)methoxy]ethyl)-4-(3-phenylpropyl)piperazine dihydrochloride) binding was performed using rat striatal membranes; no differences were found between nicotine and saline-control groups at 5, 10 or 40 min post-injection, indicating that nicotine did not increase striatal dopamine transporter density; however, [(3)H]GBR 12935 binding assays determine both cell surface and intracellular dopamine transporter. Changes in cellular dopamine transporter localization in striatum were determined using biotinylation and subfractionation approaches; no differences between nicotine and saline-control groups were observed at 10 and 40 min post-injection. These results suggest that the nicotine-induced increase in dopamine uptake and clearance in striatum may occur via a trafficking-independent mechanism.

Nornicotine, a major tobacco alkaloid and nicotine metabolite, accumulates in rat brain in pharmacologically relevant concentrations following repeated nicotine administration. Nornicotine-evoked striatal dopamine release is Ca2+-dependent, stereoselective and sensitive to nicotinic receptor antagonists, indicating nicotinic receptor-mediation. The present study determined if S-(-)-nornicotine desensitizes nicotinic receptors and if cross-desensitization to S-(-)-nicotine occurs. S-(-)-Nicotine (10 and 100 nM) diminished [3H]overflow from [3H]dopamine-preloaded rat striatal slices following subsequent superfusion with 10 microM S-(-)-nicotine (46% and 74%, respectively) or 10 microM S-(-)-nornicotine (59% and 81%, respectively). S-(-)-Nornicotine (1 and 10 microM) diminished the response to subsequent superfusion with 10 microM S-(-)-nornicotine (85% and 97%, respectively) or 10 microM S-(-)-nicotine (82% and 88%, respectively). Thus, similar to S-(-)-nicotine, S-(-)-nornicotine desensitizes nicotinic receptors. but with approximately 12-fold lower potency. Cross-desensitization suggests involvement of common nicotinic receptor subtypes. Therefore, S-(-)-nicotine metabolites, such as nornicotine, have neuropharmacologically relevant effects.

The current study examined the effect of the novel nicotinic acetylcholine receptor antagonist, N,N′-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB), on nicotine-induced dopamine metabolism in rat nucleus accumbens, striatum and medial prefrontal cortex. Acute nicotine (0.5 mg/kg, s.c.) produced an increase in the content of dihydroxyphenylacetic acid (DOPAC) in nucleus accumbens, but not in striatum or medial prefrontal cortex. Pretreatment with bPiDDB (1 or 3 mg/kg, s.c.) dose-dependently inhibited the nicotine-induced increase in DOPAC content in nucleus accumbens. These results indicate that bPiDDB inhibits the nicotine-induced increase in DOPAC in reward-relevant brain region targeting nicotinic acetylcholine receptors.

In addition to S(-)-nicotine, several minor tobacco alkaloids ((+/-)-nornicotine, anabaseine, S(-)-anabasine, and S(-)-N-methylanabasine) are present in tobacco smoke. This study demonstrates that these alkaloids increase fractional 3H release in a concentration-dependent manner from rat striatal slices preloaded with [3H]dopamine, with desensitization of this response. The rank order of EC50 values was S(-)-nicotine (3.0 +/- 2.2 microM)(+/-)-nornicotine (6.7 +/- 2.1 microM)anabaseine (15.4 +/- 6.1 microM) = S(-)-N-methylanabasine (16.3 +/- 4.7 microM) = S(-)-anabasine (19.3 +/- 3.2 microM). The alkaloids did not modulate fractional 3H release evoked by electrical-field depolarization. Thus, minor tobacco alkaloids may contribute to the apparent neuroprotective effects of smoking in neurodegenerative diseases.

Substrates for cytochrome P450-2D1 exhibit a high affinity for sigma binding sites suggesting that sigma sites may be associated with the cytochrome P450-2D1 isozyme. In contrast to Sprague-Dawley, Dark Agouti rat liver does not express the P450-2D1 gene product. Therefore, if a subpopulation of sigma sites is associated with the P450-2D1 enzyme, then the number (Bmax) of sigma sites is predicted to be decreased in Dark Agouti brain and liver compared to Sprague-Dawley tissues. In the present study, binding of [3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ([3H](+)3-PPP) in brain and liver from Dark Agouti, Sprague-Dawley, Long Evans and Wistar rat strains was examined. Results demonstrate marked variation in Bmax among the strains, with a consistently lower value for Dark Agouti tissues. However, the absolute difference in sigma binding between brain and liver for each strain was not consistent with reported differences in the activity or levels of P450-2D1. Additionally, the percentage decrease in Bmax for Dark Agouti liver was found to be similar to that for Dark Agouti brain. Taken together these results suggest that P450-2D1 does not account for the strain-related difference in sigma binding; but rather, other genetic factor(s) may be responsible for the decrease in the number of sigma sites in the Dark Agouti strain compared to the other rat strains examined.

The involvement of α 2 -noradrenergic receptors in the expression of opiate withdrawal was studied using an operant behavioral model of acute morphine dependence. Clonidine, an α 2 -agonist, attenuated and yohimbine, an α 2 -antagonist, exacerbated the naloxone-induced suppression of fixed ratio 15 responding in rats pretreated several hours earlier with a single, moderate dose of morphine. These data indicate that the α 2 -agonist action of clonidine is responsible for its amelioration of withdrawal symptoms. It also validates the acute dependence model for studying the pharmacology of opiates and associated adaptive processes.